Polyalkylene glycol derivative and modified bio-related substance

ABSTRACT

A polyalkylene glycol derivative containing a compound of the formula (1):  
                 
 
wherein R is a hydrocarbon group having 1 to 24 carbon atoms, OA 2  is an oxyalkylene group having 2 to 4 carbon atoms, the groups represented by R are the same or different from each other in one molecule, the groups represented by OA 2  are the same or different from each other in one molecule, m is an average number of moles of the oxyalkylene group added, m represents 10 to 1000, and X represents a functional group capable of chemically reactive with a bio-related substance, 
polydispersity Mw/Mu of the above polyalkylene glycol derivative in gel permeation chromatography satisfying the following relationship: 
 
 Mw /Mn≦1.07 
wherein Mw represents a weight average molecular weight and Mn represents a number average molecular weight.

This is a Continuation-In-Part application which claims priority under35 U.S.C §120 to application Ser. No. 10/716,432 filed in the UnitedStates on Nov. 20, 2003; the entire content of which is herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a reactive polyalkylene glycolderivative, and a bio-related substance modified with the reactivepolyalkylene glycol derivative.

BACKGROUND ART OF THE INVENTION

Recently, a large number of proteins, polypeptides, synthetic compounds,and compounds extracted from natural resources having physiologicalactivity have been found out and the application thereof topharmaceuticals has been extensively studied. However, thesephysiologically active substances have short half-lives in blood whenthey are injected to a body and hence it is difficult to obtain asufficient pharmacological effect. This is because the physiologicallyactive substances injected to a body are usually cleared from the bodybecause of the filtration through glomeruli in the kidney and the uptakeby macrophages in the liver, spleen, and the like. Therefore, it isattempted to improve the behavior in a body by including thesephysiologically active substances in liposomes or polymer micelles orincreasing their molecular weight through chemical modification withpolyethylene glycol which is an amphiphatic polymer. Polyethylene glycolexhibits a low interaction with the other bio-components owing to itssteric repulsion effect and as a result, proteins and polypeptides suchas enzymes modified with polyethylene glycol exhibit an effect ofavoiding the filtration through glomeruli in the kidney andbio-reactions such as immunoreaction, so that they achieve half-lives inblood longer than those of unmodified substances. Moreover, they alsohave decreased toxicity and antigenicity and further exhibit an effectof enhancing the solubility of a sparingly water-soluble compound havinga high hydrophobicity.

Hitherto, in the case of modifying a physiologically active substancewith polyethylene glycol, particularly in the case of modifying alow-molecular-weight drug or peptide, there arises a problem that fewnumbers of reactive functional groups can be used for the modificationwith polyethylene glycol. Furthermore, when a peptide or drug ismodified with many polyethylene glycol molecules for obtaining asufficient effect of the modification with polyethylene glycol, theactive site of the peptide or drug is blocked and hence problems mayarise that its own function and efficacy cannot be exhibitedsufficiently and enough solubility in water cannot be obtained.

For solving such problems, the reduction of the number of modificationwith polyethylene glycol using a branched polyethylene glycol derivativehas been attempted. JP-B-61-42558 proposes a polyethyleneglycol-modified L-asparaginase. However, cyanuric chloride as a startingmaterial for a reactive polyethylene glycol derivative has threereactive sites and hence it is difficult to introduce two polyethyleneglycol chains thereinto selectively. Accordingly, it is difficult tosynthesize a highly pure polyethylene glycol-modified L-asparaginase.

Also, JP-A-10-67800 proposes a polyethylene glycol-modified interferonα. However, this substance has three urethane and amide bonds includingthe linkage between interferon a and the poly(ethylene glycol)oxy group.These bonds are labile to hydrolysis during storage or during thereaction under an alkaline condition and as a result, there arises aproblem that the branched polyethylene glycol moiety is decomposed intoa single chain. This is because the polyethylene glycol derivative whichis the intermediate material has been produced by a method wherein twomonomethoxypolyethylene glycols and amino groups at α- and ε-positionsof lysine are combined through urethane bonds and then the carboxylresidue of lysine is converted into a succinimide ester. Moreover, inorder to produce the polyethylene glycol-modified interferon α, therearises a problem that increased impurities are produced owing to themulti-step process, such as the activation of the terminal hydroxylgroups of two monomethoxypolyethylene glycols, the combination withlysine, the activation of the carboxyl residue of lysine, and thecombination with interferon α.

Accordingly, it is desired to develop a bio-related substance formed byhighly stable bonds, a process for producing the same, and a branchedreactive polyalkylene glycol derivative which can be produced in aconvenient manner and in a high purity and has a higher stability.

SUMMARY OF THE INVENTION

A first object of the invention is to provide a polyalkylene glycolderivative having a reactive group capable of being combined with abio-related substance at the primary carbon at the 1-position of theglycerin skeleton and having polyalkylene glycol chains at the 2- and3-positions, wherein all the bonds except the bonding site with thebio-related substance is formed by ether bonds having a high stability.

A second object of the invention is to provide a bio-related substancehaving a branched poly(alkylene glycol)oxy group which is formed bystable bonds and is hardly decomposed into a single chain.

As a result of extensive studies for solving the above problems, thepresent inventors have found a novel branched polyalkylene glycolderivative and a novel bio-related substance having a branchedpoly(alkylene glycol)oxy group, and thus accomplished the invention.

Namely, the invention relates to a polyalkylene glycol derivativecomprising a compound of the following formula (1):

wherein R is a hydrocarbon group having 1 to 24 carbon atoms, OA² is anoxyalkylene group having 2 to 4 carbon atoms, the groups represented byR are the same or different from each other in one molecule, the groupsrepresented by OA² are the same or different from each other in onemolecule, m is an average number of moles of the above oxyalkylene groupadded, m represents 10 to 1000, and X represents a functional groupcapable of chemically reacting with a bio-related substance,

polydispersity Mw/Mn of the above polyalkylene glycol derivative in gelpermeation chromatography satisfying the following relationship:Mw/Mn≦1.07wherein Mw represents a weight average molecular weight and Mnrepresents a number average molecular weight.

The branched polyalkylene glycol derivative of the invention can providea polyalkylene glycol derivative having a reactive group capable ofbeing combined with a bio-related substance at the primary carbon at the1-position of the glycerin skeleton and having polyalkylene glycolchains at the 2- and 3-positions, wherein all the bonds except thebonding site with the bio-related substance is formed by ether bondshaving a high stability. The modified bio-related substance of theinvention is formed by stable bonds and is hardly decomposed into asingle chain.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a model chart of a chromatogram obtained by gel permeationchromatography of a polyalkylene glycol derivative.

FIG. 2 is a model chart of a chromatogram obtained by gel permeationchromatography of a polyalkylene glycol derivative.

FIG. 3 is a model chart of a chromatogram obtained by liquidchromatography using an ion-exchange column.

FIG. 4 is a chart illustrating a result of GPC measurement before anaccelerated aging test of the compound (p-19).

FIG. 5 is a chart illustrating a result of GPC measurement after anaccelerated aging test of the compound (p-19).

FIG. 6 is a chart illustrating a result of GPC measurement before anaccelerated aging test of the compound (p-21).

FIG. 7 is a chart illustrating a result of GPC measurement after anaccelerated aging test of the compound (p-21).

FIG. 8 is a result of electrophoresis of the compounds obtained bymodifying Humanin with the compound (p9) and the compound (p25).

DETAILED DESCRIPTION OF THE INVENTION

The polyalkylene glycol derivative of the present invention isrepresented by the formula (1):

wherein R is a hydrocarbon group having 1 to 24 carbon atoms, OA² is anoxyalkylene group having 2 to 4 carbon atoms, the groups represented byR are the same or different from each other in one molecule, the groupsrepresented by OA² are the same or different from each other in onemolecule, m is an average number of moles of the above oxyalkylene groupadded, m represents 10 to 1000, and X represents a functional groupcapable of chemically reacting with a bio-related substance,polydispersity Mw/Mn of the above polyallylene glycol derivative in gelpermeation chromatography satisfying the following relationship:Mw/Mn≦1.07wherein Mw represents a weight average molecular weight and Mnrepresents a number average molecular weight.

R in the formula (1) is a hydrocarbon group having 1 to 24 carbon atomsand specific hydrocarbon groups include hydrocarbon groups such as amethyl group, an ethyl group, a propyl group, an isopropyl group, abutyl group, a tert-butyl group, a pentyl group, an isopentyl group, ahexyl group, a heptyl group, a 2-ethylhexyl group, an octyl group, anonyl group, a decyl group, an undecyl group, a dodecyl group, atridecyl group, a tetradecyl group, a pentadecyl group, a hexadecylgroup, a heptadecyl group, an octadecyl group, an oleyl group, anonadecyl group, an eicosyl group, a heneicosyl group, docosyl group, atricosyl group, a tetracosyl group, a benzyl group, a cresyl group, abutylphenyl group, and a dodecylphenyl group. The hydrocarbon group ispreferably a hydrocarbon group having 1 to 10 carbon atoms, morepreferably a methyl group or an ethyl group, further preferably a methylgroup. Rs may be the same or different from each other in one molecule.

OA² represents an oxyalkylene group having 2 to 4 carbon atoms.Specifically, it includes an oxyethylene group, an oxypropylene group,an oxytrimethylene group, an oxy-1-ethylethylene group, anoxy-1,2-dimethylethylene group, and an oxytetramethylene group. Theoxyalcylene groups may be the same or different from each other and maybe added randomly or block-wise. In general, the fewer the carbon atomsare, the higher the hydrophilicity is. The group is preferably anoxyethylene group or an oxypropylene group, more preferably anoxyethylene group. OA²s may be the same or different from each other inone molecule. m is an average number of moles of the oxyalkylene groupadded. m represents 10 to 1000, preferably 20 to 1000, more preferably50 to 1000, most preferably 100 to 800.

X is not particularly limited as far as it is a functional group capableof chemically reacting with a bio-related substance or an unsaturatedbond. In a preferable embodiment, X is a group represented by the group(I):

The groups represented by (c), (d), (h), and (j) are preferable in thecase of reaction with an amino group of the bio-related substance, thegroups represented by (b), (c), (d), (e), (h), (i), and (j) in the caseof reaction with a mercapto group of the bio-related substance, thegroup represented by (i) in the case of reaction with an unsaturatedbond of the bio-related substance, and the groups represented by (a) and(i) in the case of reaction with a carboxyl group of the bio-relatedsubstance. Moreover, the groups represented by (a), (f), (g), and (i)are preferable in the case of reaction with an aldehyde group of thebio-related substance. In the case that the bio-related substance doesnot have an amino group, a mercapto group, an unsaturated bond, acarboxyl group, or an aldehyde group, these groups may be suitablyintroduced thereto.

Z in the group (I) is a linker between the reactive functional group andthe carbon at 1-position of glycerin and is not particularly limited asfar as it is a covalent bond but preferably includes an alkylene groupalone or an alkylene group containing an ester bond, a urethane bond, anamide bond, an ether bond, a carbonate bond, or a secondary amino group.Preferable alkylene group includes a methylene group, an ethylene group,a trimethylene group, a propylene group, an isopropylene group, atetramethylene group, a butylene group, an isobutylene group, apentamethylene group, and a hexamethylene group. More preferable is astructure of the following (z1). Further preferable as an alkylene groupcontaining an ester bond is a structure of the following (z2). Morepreferable as an alkylene group containing an amide bond is a structureof the following (z3). A structure of the following (z4), (z7), or (z8)is more preferable as an alkylene group containing an ether bond. Morepreferable as an alkylene group containing a urethane bond is astructure of the following (z5). A structure of the following (z6) ismore preferable as an alkylene group containing a secondary amino group.In each formula, s is an integer of 1 to 6, preferably an integer of 1to 5.

W¹ in the group (I) is a halogen atom selected from Cl, Br, and I, andpreferable is a case of I.

FIG. 1 is a model chart of a chromatogram obtained by gel permeationchromatography of a polyalkylene glycol derivative.

In the polyalkylene glycol derivative of the formula (1), polydispersityMw/Mn in gel permeation chromatography indicates polydispersity Mw/Mn inthe whole peaks from elution start point to elution end point andsatisfies a relationship:Mw/Mn≦1.07.More preferable is the case wherein it satisfies a relationship:Mw/Mn≦1.06.Further preferable is the case wherein it satisfies a relationship:Mw/Mn≦1.05.

The case that Mw/Mn is larger than 1.07 means that high-molecular-weightimpurities and low-molecuar-weight impurities are present in largeamounts and when a bio-related substance is combined, physicalproperties becomes heterogeneous, so that the product is not preferableas a pharmaceutical.

In the invention, at gel permeation chromatography, LC10AVP was employedas a GPC system and measurement was carried out under the followingconditions:

-   developing solvent: DMF (containing 10 mM lithium bromide); flow    rate: 0.7 ml/min; column: PLgel MIXED-D two columns; column    temperature: 65° C.; detector: RI (manufactured by shodex); sample    amount: 1 mg/mL, 100 μl.

Moreover, at analysis of an aldehyde compound, since an associate isformed under the above conditions and thus it is difficult to conductcorrect measurement of molecular weight distribution, measurement wascarried out under the following conditions:

-   system: Alliance2695 (Nihon Waters K.K.); developing solvent: 100 mM    sodium acetate buffer solution (pH=5.2, containing 0.02% NaN₃); flow    rate: 0.5 ml/min; column: Ultlahydrogel500+Ultlahydrogel250 2    columns; column temperature: 30° C.; detector: RI; sample amount: 5    mg/mL, 20 μl.

FIG. 2 is a model chart of a chromatogram obtained by gel permeationchromatography of a polyalkylene glycol derivative.

In the polyalkylene glycol derivative of the formula (1), thelow-molecular-weight impurities (%) is represented by(Area-L/Area-A)×100 where a peak showing a maximum point of refractiveindex is regarded as a main peak among peaks excluding peaksattributable to the developing solvent and the like used and pseudopeaks owing to fluctuation of base line originated from the columns andapparatus used, a line connecting an elution start point and an elutionend point of a chromatogram is used as a base line, a total peak area onand above the base line is defined as Area-A, and a peak area from aminimum point between a peak top of the main peak and a peak top of apeak to be observed next to the main peak to the elution end point isdefined as Area-L. In the case that any minimum point between the peaksis not observed, a peak area from an inflection point to be firstobserved starting from the peak top of the main peak to the elution endpoint is defined as Area-L.

In the polyalkylene glycol derivative of the formula (1) of theinvention, Area-A and Area-L preferably satisfy a relationship:(Area-L/Area-A)×100≦6(%),more preferably satisfy a relationship:(Area-L/Area-A)×100≦5(%),further preferably satisfy a relationship:(Area-L/Area-A)×100≦4(%).

The case that (Area-L/Area-A)×100 is larger than 6(%) means thatlow-molecular-weight impurities are present in a large amount and, whena bio-related substance is combined, physical properties becomesheterogeneous, so that the product is not preferable as apharmaceutical.

The modified bio-related substance of the invention represents acombined product of the compound of the formula (1) with a bio-relatedsubstance.

The “bio-related substance” according to the invention means a substancerelating to a body. The substances relating to a body include thefollowing.

(1) Animal Cell-Constituting Materials Such as Phospholipids,Glycolipides, and Glycoproteins

The animal cell-constituting materials are components constituting cellmembranes or the like and the kind is not particularly limited butexamples thereof include phospholipids, glycolipides, and glycoproteins.Examples of more specific phospholipids include phosphatidic acid,phosphatidylcholine, phosphatidylethanolamine, cardiolipin,phosphatidylserine, and phosphatidylinositol. In addition, lyso isomersthereof are also included. These phospholipids may be those derived fromnatural products such as egg yolk or soybean or may be synthesizedproducts. The composition of fatty acids is not particularly limited butmay include fatty acids having 12 to 22 carbon atoms. These fatty acidsmay be saturated fatty acids or may be those containing an unsaturatedbond. Examples of more specific glycolipids include ceramides,cerebrosides, sphingosines, gangliosides, and glyceroglycolipids. Inaddition, fatty acids, monoglycerides, diglycerides, cholesterols, andbile acid are also included.

(2) Body Fluid-Constituting Substances Such as Blood, Lymph, and BoneMarrow Liquid

The body fluid-constituting substances mean fluid components existinginside and outside cells and the kind is not particularly limited butexamples thereof include blood, lymph, and bone marrow liquid. Examplesof more specific body fluid-constituting components include hemoglobin,albumin, and blood coagulation factors.

(3) Physiologically Active Substances Such as Vitamins,Neurotransmitters, Proteins, Polypeptides, and Drugs

The physiologically active substances mean components controlling bodyfunctions and the kind is not particularly limited but examples thereofinclude vitamins, neurotransmitters, proteins, polypeptides, and drugs.

Examples of more specific vitamins include vitamin A, vitamin B, vitaminC, vitamin D, vitamin E, and vitamin K.

Examples of more specific neurotransmitters include adrenalin,noradrenalin, dopamine, acetylcholine, GABA, glutamic acid, and asparticacid.

Examples of more specific proteins and polypeptides include thefollowing. Hormones such as neurohypophysial hormone, thyroid hormone,male sex hormone, female sex hormone, and adrenal cortex hormone. Serumproteins such as hemoglobin and blood factors. Immunoglobulins such asIgG, IgE, IgM, IgA, and IgD. Cytokines and fragments thereof, such asinterleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL,11 and IL-12 subtypes), interferons (−α, −β, −γ),granulocyte-colony stimulating factors (α and β types),macrophage-colony stimulating factor, granulocyte-macrophage colonystimulating factor, platelet-derived growth factor,phospholipase-activating protein, insulin, glucagon, lectin, ricin,tumor necrosis factor, epidermal growth factor, transforming growthfactors (−α, −β), fibroblast growth factor, hepatocyte growth factor,vascular endothelial growth factor, nerve growth factor, bone growthfactor, insulin-like growth factor, heparin binding growth factor, tumorgrowth factor, glial cell line-derived neurotrophic factor, macrophagedifferentiating factor, differentiation-inducing factor, leukemiainhibitory factor, amphiregurin, somatomedin, erythropoietin,hemopoietin, thrombopoietin, and calcitonin. Enzymes such as proteolyticenzymes, oxidoreductases, transferases, hydrolases, lyases, isomerases,ligases, asparaginases, arginases, arginine deaminases, adenosinedeaminases, superoxide dismutases, endotoxinases, catalases,chymotrypsin, lipases, uricases, elastases, streptokinases, urokinases,prourokinases, adenosine diphosphatases, tyrosinases, bilirubinoxidases, glucose oxidases, glucodases, glactosidases,glucocerebrosidases, and glucouronidases. Monoclonal and polyclonalantibodies and fragments thereof polyamino acids such as poly-L-lysineand poly-D-lysine. Vaccines such as hepatitis B vaccine, malariavaccine, melanoma vaccine, and HIV-1 vaccine, and antigens. In addition,glycoproteins are also included. Furthermore, also included arestructurally similar substances having physiological activity similar tothat of these physiologically active substances.

Moreover, these proteins and polypeptides may be isolated from naturalsources thereof or cells subjected to genetic engineering or may beproduced via various synthetic processes.

The drugs are not particularly limited but more preferably includeanticancer drugs and antifungal drugs.

More specific anticancer drugs are not particularly limited but, forexample, include paclitaxel, adriamycin, doxorubicin, cisplatin,daunomycin, mitomycin, vincristine, epirubicin, methotrexate,5-fluorouracil, aclacinomycin, idamycin, bleomycin, pirarubicin,peplomycin, vancomycin, and camptothecine.

Specific antifungal drugs are not particularly limited but, for example,include amphotericin B, nystatin, flucytosine, miconazole, fluconazole,itraconazole, ketoconazole, and peptide antifungal drugs.

Moreover, these physiologically active substances also includeflavonoids, terpenoids, carotenoids, saponins, steroids, quinones,anthraquinones, xanthones, coumarins, alkaloids, porphyrins, andpolyphenols, which possess, for example, antioxidant action, PAFinhibitory action, antiinflammatory action, and antifungal action.

The number of modifications with the polyalkylene glycol derivative tothe bio-related substance is not particularly limited but is preferably1 to 100, more preferably 1 to 20.

Tables 1 and 2 show relation between a residual group T of the abovebio-related substance and a functional group X which forms a chemicalbond with the residual group T. TABLE 1 Reactive group ofphysiologically active substance X group T—NH₂(amino group) T—SH(Mercapto group)

(a) —Z—NH₂

TABLE 2 Reactive group of physiologically active substance X groupT—NH₂(Amino group) T—SH (Mercapto group)

(g) —Z—ONH₂

—Z—ON═CH—T (oxime) (h) —Z—COOH

(j) —Z—SH

—Z—S—S—T (disulfide)

As is apparent from the tables, the functional group X in thepolyalkylene glycol derivative of the invention and the bio-relatedsubstance are combined by, for example, an amide bond, a secondary aminogroup, a urethane bond, a thioester bond, a sulfide bond, a disulfidebond, a thiocarbonate bond, an oxime, a hydrazone, or a thioacetal bond.

The modified bio-related substances of the invention can be produced asfollows.

(Case of Reacting an Amino Group of a Bio-Related Substance With aPolyalkylene Glycol Derivative of the Invention)

In the case of the modification with an amino group of a bio-relatedsubstance, the compounds (c), (d), (h), and (j) of the invention areused. More preferably, (c), (d), and (j) are used. At the reaction, thecompounds (c), (d), (h), and (j) of the invention may be reacted in aratio of equimolar or more to the bio-related substance. The reactionsolvent is not particularly limited as far as it does not participate inthe reaction, but in the case of reacting a protein or polypeptide,preferable solvents include buffer solutions such as phosphate buffersolutions, borate buffer solutions, Tris-acid buffer solutions, acetatebuffer solutions, and carbonate buffer solutions. Furthermore, anorganic solvent which does not deactivate the protein or polypeptide anddoes not participate in the reaction, such as acetonitrile, dimethylsulfoxide, dimethylformamide, or dimethylacetamide, may be added. In thecase of reacting an anticancer drug, antifungal drug, or phospholipid,preferable solvents include, in addition to the above buffer solutions,toluene, benzene, xylene, acetonitrile, ethyl acetate, diethyl ether,t-butyl methyl ether, tetrahydrofuran, chloroform, methylene dichloride,dimethyl sulfoxide, dimethylformamide, dimethylacetamide, water,methanol, ethanol, n-propanol, 2-propanol, and n-butanol. Also, thesolvent need not be used. The order of adding the polyalkylene glycolderivative of the invention and the bio-related substance is optional.The reaction temperature is not particularly limited as far as it doesnot deactivate the bio-related substance, but the temperature ispreferably 0 to 40° C. in the case of reacting a protein or polypeptideand is preferably −20 to 150° C. in the case of reacting an anticancerdrug, antifungal drug, or phospholipid. The reaction time is preferably0.5 to 72 hours, more preferably 1 to 24 hours. At the reaction, acondensing agent such as N,N′-dicyclohexylcarbodilmide (DCC) or1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) may beused. In the case that a Schiff base is formed by the reaction, it maybe subjected to reduction treatment using a reducing agent such assodium cyanoborohydride. A covalent bond is formed between thebio-related substance and the polyalkylene glycol derivative of theinvention by carrying out the reaction. An amide bond is formed in thecase of using (c) or (h), and a urethane bond is formed in the case ofusing (d). A Schiff base is formed in the case of using (j) and theSchiff base is reduced to form a secondary amino group. After completionof the reaction, the product may be purified by a purifying means suchas dialysis, salting-out, ultrafiltration, ion-exchange chromatography,electrophoresis, extraction, recrystallization, adsorption treatment,reprecipitation, column chromatography, or supercritical extraction.

(Case of Reacting a Mercapto Group of a Bio-Related Substance With aPolyalkylene Glycol Derivative of the Invention)

In the case of the modification with a mercapto group of a bio-relatedsubstance, the polyalkylene glycol derivatives of the invention (b),(c), (d), (e), (h), (i), and (j) of the invention are used. Morepreferably, (b) and (e) are used. The reaction solvent, reactionconditions, and the like are the same as in the case of using an aminogroup. At the reaction, a radical initiator such as iodine or AIBN maybe used. A covalent bond is formed between the bio-related substance andthe polyalkylene glycol derivative of the invention by carrying out thereaction, and a thioether bond is formed in the case of using (c) or(h), a thiocarbonate bond in the case of using (d), a disulfide bond inthe case of using (i), a sulfide bond in the case of using (b) or (e),and a thioacetal bond is formed in the case of using (j).

(Case of Reacting an Unsaturated Bond of a Bio-Related Substance With aPolyalkylene Glycol Derivative of the Invention)

In the case of the modification with an unsaturated bond of abio-related substance, the polyalkylene glycol derivative (i) of theinvention is used. The reaction solvent, reaction conditions, and thelike are the same as in the case of using an amino group. At thereaction, a radical initiator such as iodine or AIBN may be used. Asulfide bond is formed between the bio-related substance and thepolyalkylene glycol derivative of the invention by carrying out thereaction.

(Case of Reacting a Carboxyl Group of a Bio-Related Substance With aPolyalkylene Glycol Derivative of the Invention)

In the case of the modification with a carboxyl group of a bio-relatedsubstance, the polyalkylene glycol derivative of the invention (a) or(i) of the invention is used. The reaction solvent, reaction conditions,and the like are the same as in the case of using an amino group. At thereaction, a condensing agent such as DCC or EDC may be optionally used.A covalent bond is formed between the bio-related substance and thepolyalkylene glycol derivative of the invention by carrying out thereaction, and a thioester bond is formed in the case of using (i) and anamide bond in the case of using (a).

(Case of Reacting an Aldehyde Group of a Bio-Related Substance With aPolyalkylene Glycol Derivative of the Invention)

In the case of the modification with an aldehyde group of a bio-relatedsubstance, the polyalkylene glycol derivative of the invention (a), (f),(g), or (i) of the invention is used. The reaction solvent, reactionconditions, and the like are the same as in the case of using an aminogroup. In the case that a Schiff base is formed, it may be subjected toreduction treatment using a reducing agent such as sodiumcyanoborohydride. A thioacetal bond is formed in the case of using (i),a secondary amino group in the case of using (a), an oxime in the caseof using (g), and a hydrazone bond in the case of using (f).

Moreover, in the case that a bio-related substance does not have any ofan amino group, a mercapto group, an unsaturated bond, a carboxyl group,and an aldehyde group, the bio-related substance can be modified byintroducing a reactive group suitably into the bio-related substance andusing a polyalkylene glycol derivative of the invention.

The polyalkylene glycol derivatives of the formula (1) of the inventioncan be, for example, produced as follows. After the primary hydroxylgroup residue of 2,2-dimethyl-1,3-dioxolane-4-methanol is protected witha benzyl group, the cyclic acetal structure is deprotected under anacidic condition to obtain the following general formula (pa). Analkylene oxide is polymerized in an amount of 10 to 1000 mol to thenewly formed two hydroxyl groups to obtain the following general formula(pb). Then, the terminal ends are alkyl-etherified to obtain thefollowing general formula (pc). Thereafter, the benzyl group isdeprotected and thereby, the compound of the following general formula(p) wherein X is a hydroxyl group can be obtained.

As above, a highly pure branched polyalkylene glycol derivative can beproduced in high yields in an industrially suitable manner by using thealkylene oxide-addition polymerization reaction, without columnpurification.

Using the hydroxyl group of the compound (p) thus obtained, thepolyalkylene glycol derivative of the invention can be produced bymodifying hydroxy group into various reactive groups shown in the group(I).

The polyalkylene glycol derivative of the invention having eachfunctional group of the group (I) can be reacted with a bio-relatedsubstance but in some cases, the polyalkylene glycol derivative of theinvention can be further reacted with the other compound to produceother polyalkylene glycol derivative and it can be then reacted with abio-related substance. For example, using the polyalkylene glycolderivative having a functional group (a) belonging to the group (I) as astarting material, the polyalkylene glycol derivative having (b) of thegroup (I) can be synthesized. Moreover, using the polyalkylene glycolderivative having a functional group (h) belonging to the group (I) asan starting material, the polyalkylene glycol derivative having (c) ofthe group (I) can be synthesized.

The following will describe a process for producing the polyalkyleneglycol derivative of the invention.

The benzyl etherification of 2,2-dimethyl-1,3-dioxolane-4-methanol canbe carried out in the following manner.

-   1) It can be achieved by reacting benzyl chloride or benzyl bromide    with 2,2-dimethyl-1,3-dioxolane-4-methanol in an aprotic solvent or    without any solvent in the presence of an alkali catalyst such as    sodium hydroxide or potassium hydroxide.-   2) It can be achieved by converting the hydroxyl group of    2,2-dimethyl-1,3-dioxolane-4-methanol in an aprotic solvent or    without any solvent using sodium, potassium, sodium hydride,    potassium hydride, sodium methoxide, potassium methoxide, potassium    t-butoxide, or the like into an alcoholate and reacting the    alcoholate with benzyl chloride or benzyl bromide under a basic    condition.-   3) It can be achieved by activating the hydroxyl group of    2,2-dimethyl-1,3-dioxolane-4-methanol with methanesulfonyl chloride,    p-toluenesulfonyl chloride, 2,2,2-trifluoroethanesulfonyl chloride,    or the like in an aprotic solvent or without any solvent, followed    by the reaction with an alcoholate of benzyl alcohol.

The deprotection of the cyclic acetal structure which follows the benzyletherification is achieved by the reaction in an aqueous solutionadjusted to pH 1 to 4 with an acid such as acetic acid, phosphoric acid,sulfuric acid, or hydrochloric acid, whereby a compound of the formula(pa) can be produced.

The method of addition polymerization of an alkylene oxide to thecompound of the formula (pa) having two hydroxyl groups newly formed bythe deprotection of the cyclic acetal to obtain a compound of theformula (pb) is not particularly limited but can be achieved via thefollowing steps (C1) and (C2).

Step (C1): as a method of alcoholation of the compound of the formula(pa), the alcoholation is carried out using sodium or potassium,preferably sodium as a catalyst, in an catalyst amount of 5 to 50% bymol, followed by dissolution at 10 to 50° C.

Step (C2): an alkylene oxide addition polymerization is carried out at areaction temperature of 50 to 130° C.

With regard to the catalyst amount in the step (C1), since thepolymerization rate of the alkylene oxide decreases at less than 5% bymol and heat history increases to result in the formation of impuritiessuch as a terminal vinyl ether compound, the use of the catalyst in anamount of 5% by mol or more is advantageous in the production of a highquality high-molecular-weight compound. When the catalyst amount exceeds50% by mol, the viscosity of the reaction liquid increases or the liquidsolidifies at the alcoholation reaction and thus there is a tendencythat the stirring efficiency decreases and the alcoholation is notaccelerated. Moreover, when the liquid solidifies, handling thereoftends to be difficult, which causes water absorption. When thealcoholate has absorbed water, an alkylene glycol compound derived fromwater is formed and is contained as an impurity undesirable in medicaluse.

When the temperature at the dissolution is higher than 50° C., adecomposition reaction may occur to form benzyl alcohol and glycerin.When benzyl alcohol is formed, it initiates addition polymerization withthe alkylene oxide, whereby a low-molecular-weight impurity having amolecular weight 0.5 time the molecular weight of the target compound.When the low-molecular-weight impurity derived from benzyl alcohol isformed, a functional group is introduced via alkyl-etherification of thehydroxyl group and deprotection in the subsequent steps as in the caseof the target compound, so that the impurity is converted into alow-molecular-weight impurity which is reactive with a bio-relatedsubstance. There is a possibility that such impurity may react with abio-related substance and change the physical properties of theresulting preparation. Moreover, when glycerin is formed, it alsoinitiates addition polymerization with the alkylene oxide to form ahigh-molecular-weight impurity having a molecular weight 1.5 times thatof the target compound. Since the high-molecular-weight impurity doesnot have a benzyl group and its terminal hydroxyl group is onlyalkyl-etherified, no functional group is introduced. However, when thecombination with a drug or the like is carried out while such impurityis contained, the resulting preparation becomes inhomogeneous and hencethe quality tends to be varied. Also, the preparation is not suitable ina medical use where a highly pure product is required.

When the dissolution is carried out at a temperature lower than 10° C.,like the case that the catalyst amount is more than 50% by mol, theviscosity of the reaction liquid increases or the liquid solidified atthe alcoholation reaction, and handling thereof tends to be difficult,and water absorption is caused.

The reaction solvent is not particularly limited as far as it is anaprotic solvent such as toluene, benzene, xylene, acetonitrile, ethylacetate, tetrahydrofuran, chloroform, methylene dichloride, dimethylsulfoxide, dimethylformamide, or dimethylacetamide, but preferable istoluene or no solvent. The reaction time is preferably 1 to 24 hours.When the time is shorter than 1 hour, there is a possibility that thecatalyst does not completely dissolved. When the time is longer than 24hours, there is a possibility that the above decomposition reaction mayoccur.

With regard to the reaction temperature in the step (C2), when thetemperature is lower than 50° C., the polymerization rate is low andheat history increases to result in a tendency to decrease the qualityof the compound of the formula (pb). Moreover, when the temperature ishigher than 130° C., side reactions such as vinyl etherification of theterminal end occur during the polymerization and thus the quality of thetarget compound tends to decrease. During the polymerization, as themolecular weight increases, the viscosity of the reaction liquid alsoincreases, so that an aprotic solvent, preferably toluene may beoptionally added.

As another production process in the step of alcoholation, the followingstep (C3) may be mentioned.

Step (C3): Sodium methoxide, potassium t-butoxide, or potassiummethoxide, preferably sodium methoxide is added as an catalyst in anamount of 5 to 50% by mol and the reaction is carried out at 60 to 80°C. At that time, a pressure-reducing operation may be conducted in orderto facilitate the exchange reaction.

The catalyst amount is preferably 5 to 50% by mol for the reasonmentioned above. With regard to the reaction temperature, when thetemperature is lower than 60° C., the conversion of the exchangereaction decreases and alcohols such as methanol remain, which leads tothe formation of impurities having a molecular weight 0.5 time that ofthe target compound via addition polymerization of an alkylene oxide.When the temperature is higher than 80° C., a decomposition reactionoccurs. The alcoholation reaction requires elevation of the temperatureand the reaction time is desirably 1 to 3 hours since the decompositionreaction is apt to occur. When the time is shorter than 1 hour, there isa possibility that the conversion into the alcoholate decreases. Whenthe time is longer than 3 hours, a decomposition reaction may occur. Thereaction solvent is not particularly limited as far as it is an aproticsolvent, but preferable is toluene or no solvent.

The subsequent alkyl-etherification of the terminal end may be achievedby either of the following (1) or (2):

-   (1) a process of converting the terminal end of the polyalkylene    glycol chain into an alcoholate and reacting it with an alkyl    halide;-   (2) a process of activating the terminal hydroxyl group of the    polyalkylene glycol chain with methanesulfonyl chloride,    p-toluenesulfonyl chloride, 2,2,2-trifluoroethanesulfonyl chloride,    or the like, followed by the reaction with an alcoholate of an alkyl    alcohol.

Preferable is the process (2) and the following will describe it in moredetail.

The production process (2) comprises the following steps (B1), (B2), and(B3).

-   Step (B1): a step of adding a dehalogenating agent and a compound    represented by the formula (6) to a compound represented by the    formula (pb) and reacting them at 20 to 60° C. to obtain a compound    of the formula (7). At that time, each charged molar ratio satisfies    the following relationship:    Vc≧Va    Vb>Vc-   Va: number of moles of the compound represented by the formula (pb)-   Vb: number of moles of the dehalogenating agent-   Vc: number of moles of the compound represented by the formula (6).

More preferable is the case that the molar ratio satisfies the followingrelationship:20Va≧Vc≧2Va4Vc>Vb>Vc.

When Vc is smaller than 2Va, the conversion decreases and thus some ofthe hydroxyl groups in the oxyalkylene chain terminal ends remainunchanged. Thereafter, a functional group is introduced to the remaininghydroxyl group to form a polyftuctional impurity having a molecularweight the same as that of the target compound. When such apolyfunctional impurity is present, it acts as a crosslinking agent atthe combination with a bio-related substance to result in a tendency todecrease the purity of the resulting modified bio-related substance.When Vb is not larger than Vc, the conversion decreases owing toinefficient trapping of an acid which is produced as a by-product withthe progress of the reaction, so that some of the hydroxyl groups in theoxyalkylene chain terminal ends remain unchanged. Moreover, when Vc islarger than 20Va or Vb is not smaller than 4Vc, an excess amount may becontained to cause side reactions.

The dehalogenating agent to be used includes organic bases such astriethylamine, pyridine, and 4-dimethylaminopyridine, and inorganicbases such as sodium carbonate, sodium hydroxide, sodium hydrogencarbonate, sodium acetate, potassium carbonate, and potassium hydroxide.Preferable dehydrochlorinating agent is an organic base such astriethylamine, pyridine, or 4-dimethylaminopyridine.

In the compound of the formula (6) to be used, W is preferably Cl or Br,and R¹ is preferably a methyl group, a phenyl group, or a p-methylphenylgroup. More suitably, methanesulfonyl chloride where W is Cl and R¹ is amethyl group is most preferable.

The solvent to be used at that time is not particularly limited as faras it is an aprotic solvent and preferably includes toluene, benzene,xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform,methylene dichloride, dimethyl sulfoxide, dimethylformamide, ordimethylacetamide, but more preferable is toluene which enablesazeotropic removal of water in the system. The amount of the solvent tobe used at the reaction is preferably 0.5 equivalent weight to 10equivalent weight to the compound of the formula (pb). In the case thatthe compound of the formula (pb) has a large molecular weight, theviscosity of the reaction liquid increases and the conversion decreases,so that it is preferable to dilute the reaction liquid with the solvent.

The reaction temperature is not particularly limited but is preferably60° C. or lower for the purpose of inhibiting side reactions and ispreferably 20° C. or higher for the purpose of inhibiting increase ofthe viscosity of the reaction liquid. The reaction time is preferably 1to 24 hours. When the time is shorter than 1 hour, there is apossibility that the conversion is low. When the time is longer than 24hours, there is a possibility that a side reaction may occur.

At the reaction, the operation of removing water from the startingmaterials, such as azeotropic removal of water may be carried out priorto the reaction. Moreover, an antioxidant such as2,6-di-tert-butyl-p-cresol (BHT) may be added. Furthermore, a salt isformed with the progress of the reaction and the formation of thecompound of the formula (7), but the reaction mixture may be used in thesubsequent step as it is, or the salt may be removed by filtration, orafter the filtration, the compound of the formula (7) may be purified bya purification means such as extraction, recrystallization, adsorptiontreatment, reprecipitation, column chromatography, or supercriticalextraction.

Step (B2): a step of adding a compound represented by the formula (8) tothe compound of the formula (7) and reacting them at 20 to 80° C. toobtain the compound of the formula (pc). At that time, each chargedmolar ratio satisfies the following relationship:Vd>Vc

-   Vd: number of moles of the compound represented by the formula (8).

More preferable is the case that the relationship:10Vc>Vd>Vcis satisfied.R—OM   (8)

In the formula (8), R is as mentioned above and M is sodium orpotassium, preferably sodium.

When Vd is not larger than Vc, the alkyl-etherification does notsufficiently proceed and a reactive group such as a mesylate groupremains unchanged at the oxyalkylene chain terminal end. When a reactivegroup remains at the oxyalkylene chain terminal end, as mentioned above,a polyfunctional compound is formed and a serious side reaction iscaused at the combination with a bio-related substance. Moreover, whenVd is not smaller than 10Vc, an excess of the alcoholate may becontained to cause side reactions in the subsequent process.

The solvent to be used in the reaction is not particularly limited asfar as it is an aprotic solvent as mentioned above and is preferablytoluene. The amount of the solvent to be used at the reaction ispreferably an amount of 0.5 equivalent to 10 equivalents to the compoundof the formula (7). In the case that the compound of the formula (7) hasa large molecular weight, the viscosity of the reaction liquidincreases, so that it is preferable to dilute the reaction liquid withthe solvent.

The reaction temperature is not particularly limited but is preferably80° C. or lower for the purpose of inhibiting side reactions and ispreferably 20° C. or higher for the purpose of inhibiting increase ofthe viscosity of the reaction liquid. The reaction time is preferably 1to 24 hours. When the time is shorter than 1 hour, there is apossibility that the conversion is low. When the time is longer than 24hours, there is a possibility that a side reaction occurs. At thereaction, an operation of removing water from the starting materials,such as azeotropic removal of water may be carried out prior to thereaction.

Step (B3): a step of filtrating the reaction liquid or washing thereaction liquid with an aqueous inorganic salt solution having aconcentration of 10% by weight or more.

In the step, the inorganic salt is not particularly limited but ispreferably sodium chloride. When the concentration is less than 10% byweight, the target compound migrates into an aqueous layer to decreasethe process yield remarkably. The operation of washing with water may berepeated several times. The step (B3) is carried out for removingstarting materials excessively added and salts produced as by-products.The omission of the step may cause side reactions in the case that thesteps (B1) to (B3) are again carried out in the next place. In the casethat a debenzylation step is carried out as a next step, theseimpurities act as catalyst poisons and thus the conversion may beaffected.

Moreover, in order to enhance the ratio of alkyl-etherification of theoxyalkylene chain terminal end, it is preferable to repeat the steps(B1) to (B3) again. When the ratio of alkyl-etherification of theoxyalkylene chain terminal end is low, as mentioned above, there is apossibility of forming a polyfunctional impurity.

The compound of the formula (pc) thus obtained may be purified by apurification means such as extraction, recrystalization, adsorptiontreatment, reprecipitation, column chromatography, or supercriticalextraction.

The production of the compound (p) by successive debenzylation is notparticularly limited but it can be produced by hydrogenation of thefollowing step (A) using a hydrogenative reduction catalyst and ahydrogen donor.

Step (A): a step of subjecting the compound represented by the formula(pc) to a hydrogenative reduction reaction under the condition that thewater content in the reaction system is 1% or less. When the watercontent in the reaction system is more than 1%, the decompositionreaction of the polyoxyalkylene chain occurs. Since polyalkylene glycolformed by the decomposition has a hydroxyl group, it is functionalizedin the next step to form a reactive low-molecular-weight impurity. Sucha reactive low-molecular-weight impurity reacts with a bio-relatedsubstance as mentioned above and thus tends to change the properties ofthe resulting preparation.

The hydrogenative reduction catalyst is preferably palladium. Thesupport is not particularly limited but is preferably alumina or carbon,more preferably carbon. The amount of palladium is preferably 1 to 20%by weight based on the compound of the formula (4). When the amount isless than 1% by weight, the conversion of deprotection decreases andthus there is a possibility that the ratio of functionalization in thenext step decreases. Moreover, when the amount is more than 20% byweight, the decomposition reaction of the polyalkylene glycol chain mayoccur and there is a possibility that the above reactivelow-molecular-weight compound is produced as a by-product. The reactionsolvent is not particularly limited as far as the water content in thereaction system is less than 1%, but preferably includes methanol,ethanol, 2-propanol, and the like and more preferable is methanol. Thehydrogen donor is not particularly limited but include hydrogen gas,cyclohexene, 2-propanol, and the like. The reaction temperature ispreferably 40° C. or lower. When the temperature is higher than 40° C.,the decomposition reaction of the polyalkylene glycol chain may occurand there is a possibility that the reactive low-molecular-weightcompound is produced as a by-product. The reaction time is notparticularly limited. When large amount of the catalyst is used, thereaction is completed within a short period of time. But, when theamount is small, a longer period of time is required. In general, thereaction time is preferably 1 to 5 hours. When the time is shorter than1 hour, there is a possibility that the conversion is low. When it islonger than 5 hours, the decomposition reaction of the poly(alkyleneglycol) may occur.

The resulting compound of the formula (p) may be purified by apurification means such as extraction, recrystallization, adsorptiontreatment, reprecipitation, column chromatography, or supercriticalextraction.

The thus obtained compound is a polyalkylene glycol derivativerepresented by the following formula (p) and containing substantially nosecondary hydroxyl group:

wherein R is a hydrocarbon group having 1 to 24 carbon atoms, OA² is anoxyalkylene group having 2 to 4 carbon atoms, the groups represented byR are the same or different from each other in one molecule, the groupsrepresented by OA² are the same or different from each other in onemolecule, m is an average number of moles of the above oxyalkylene groupadded, and m represents 10 to 1000.

Since the compound of the formula (p) contains substantially nosecondary hydroxyl group, the conversion of the subsequent functionalgroup-introducing reaction is high and a highly pure polyalkylene glycolderivative can be obtained. In the case that a secondary hydroxyl groupis present, the conversion of the subsequent functionalgroup-introducing reaction is low and the purity of the modifiedbio-related substance decreases, so that there may arise the problem ofcontamination of the drug or the like with an impurity.

The compound (p) thus obtained has a low content of the impuritieshaving a hydroxyl group at the terminal end of the polyoxyalkylene chainshown below.

-   (A): an impurity having a hydroxyl group and a molecular weight 0.5    time that of the compound (p), which is formed by decomposition of    the compound of the formula (pa) at the alcoholation, addition    polymerization of an alkylene oxide to the resulting benzyl alcohol,    and deprotection of benzyl group in the subsequent step;-   (B): an impurity having a remaining hydroxyl group at 2- or    3-position and a molecular weight the same as that of the compound    (p), which is formed at the alkyl-etherification of the compound of    the formula (pb);-   (C): an impurity having a hydroxyl group and a low molecular weight,    which is formed by decomposition of the polyoxyalkylene chain at the    debenzylation of the compound of the formula (pc).

When a functional group is introduced using a compound (p) containing alarge amount of impurities (A), (B), and (C), impurities of the reactivepolyalkylene glycol derivatives shown below are contained.

-   (D): a reactive polyalkylene glycol derivative having a 0.5 time    molecular weight, wherein a functional group is introduced into the    impurity shown in the above (A);-   (E): a bifunctional or trifunctional reactive polyalkylene glycol    derivative having the same molecular weight, wherein a functional    group is introduced into the impurity shown in the above (B);-   (F): a low-molecular-weight reactive polyalkylene glycol derivative,    wherein a functional group is introduced into the impurity shown in    the above (C).

The following shows the reaction pathways to the compound (p).

The following will describe the introduction of a reactive group intothe hydroxyl group of the compound (p) formed by the debenzylationreaction but the other known processes may be used for synthesis.

(Process for Producing (a))

The amine compound (a) can be obtained by adding the compound (p) toacrylonitrile or the like using an inorganic base such as sodiumhydroxide or potassium hydroxide in a solvent such as water oracetonitrile to obtain a nitrile compound and then subjecting it tohydrogenation of the nitrile group in the presence of a nickel orpalladium catalyst in an autoclave. The ratio of the inorganic base tobe used for obtaining the nitrile compound is not particularly limitedbut is preferably 0.01 to 50% by weight to the compound (p). The ratioof acrylonitrile or the like to be used is not particularly limited butis preferably 0.5 to 5 equivalent weight, more preferably 1 to 4equivalent weight to the weight of the compound (p). Moreover,acrylonitrile may be used as a solvent. The reaction temperature ispreferably −50 to 100° C., more preferably −20 to 60° C. The reactiontime is preferably 10 minutes to 48 hours, more preferably 30 minutes to24 hours. The reaction solvent in the subsequent hydrogenation reactionof the nitrile compound is not particularly limited as far as it doesnot participate in the reaction, but is preferably toluene. The ratio ofthe nickel or palladium catalyst to be used is not particularly limitedbut is 0.05 to 30% by weight, preferably 0.5 to 20% by weight to thenitrile compound. The reaction temperature is preferably 20 to 200° C.,more preferably 50 to 150° C. The reaction time is preferably 10 minutesto 48 hours, more preferably 30 minutes to 24 hours. The hydrogenpressure is preferably 2 to 10 MPa, more preferably 3 to 8 MPa.Moreover, in order to prevent dimerization, ammonia may be added to thereaction system. In the case of adding ammonia, ammonia pressure is notparticularly limited but is 0.1 to 10 MPa, more preferably 0.3 to 2 MPa.The compound formed may be purified by the aforementioned purificationmeans.

(Process for Producing (d))

By reacting the compound (p) with an organic base such as triethylamine,pyridine, or 4-dimethylaminopyridine or an inorganic base such as sodiumcarbonate, sodium hydroxide, sodium hydrogen carbonate, sodium acetate,potassium carbonate, or potassium hydroxide and any one of the compoundsrepresented by the following general formula (d1) in an aprotic solventsuch as toluene, benzene, xylene, acetonitrile, ethyl acetate, diethylether, t-butyl methyl ether, tetrahydrofuran, chloroform, methylenedichloride, dimethyl sulfoxide, dimethylformamide, or dimethylacetamideor without any solvent, the compound (d) can be synthesized. Moreover,the above organic base or inorganic base need not be used. The ratio ofthe organic base or inorganic base to be used is not particularlylimited but is preferably equimolar or more to the compound (p).Furthermore, an organic base may be used as a solvent. W in (d1) is ahalogen atom selected from Cl, Br and L and is preferably Cl. The ratioof the compounds represented by the general formula (d1) to be used isnot particularly limited but is preferably equimolar or more, morepreferably equimolar to 50 molar to the compound (p). The reactiontemperature is preferably 0 to 300° C., more preferably 20 to 150° C.The reaction time is preferably 10 minutes to 48 hours, more preferably30 minutes to 24 hours. The compound formed may be purified by apurification means such as extraction, recrystallization, adsorptiontreatment, reprecipitation, column chromatography, or supercriticalextraction.

wherein W is a halogen atom selected from Cl, Br, and I.(Process for Producing (c) and (h))

The succinimide compound (c) can be obtained by reacting the compound(p) with a dicarboxylic acid anhydride such as succinic anhydride orglutaric anhydride to obtain a carboxyl compound (h), followed bycondensation with N-hydroxysuccinimide in the presence of a condensingagent such as DCC or EDC. The reaction of the compound (p) with adicarboxylic acid anhydride is carried out in the aforementioned aproticsolvent or without any solvent. The ratio of the dicarboxylic acidanhydride to be used is not particularly limited but is preferablyequimolar or more, more preferably equimolar to 5 molar to the compound(p). The reaction temperature is preferably 0 to 200° C., morepreferably 20 to 150° C. The reaction time is preferably 10 minutes to48 hours, more preferably 30 minutes to 12 hours. In the reaction, anorganic base such as triethylamine, pyridine, or dimethylaminopyridineor an inorganic base such as sodium carbonate, sodium hydroxide, sodiumhydrogen carbonate, sodium acetate, potassium carbonate, or potassiumhydroxide may be used as a catalyst. The ratio of the catalyst to beused is preferably 0.1 to 50% by weight, more preferably 0.5 to 20% byweight. The carboxyl compound (h) thus formed may be purified by theaforementioned purification means or may be used as it is in the nextcondensation reaction.

The subsequent condensation reaction is also carried out in theaforementioned aprotic solvent or without any solvent. The condensingagent is not particularly limited but is preferably DCC. The ratio ofDCC to be used is preferably equimolar or more, more preferablyequimolar to 5 molar to the compound (h). The ratio ofN-hydroxysuccinimide to be used is preferably equimolar or more, morepreferably equimolar to 5 molar to the compound (h). The reactiontemperature is preferably 0 to 100° C., more preferably 20 to 80° C. Thereaction time is preferably 10 minutes to 48 hours, more preferably 30minutes to 12 hours. The compound formed may be purified by theaforementioned purification means.

Moreover, the compound (c) can be, for example, produced by thefollowing process. It can be obtained by reacting the compound (p) withN,N′-disuccinimidyl carbonate. The reaction of the compound (p) withN,N′-disuccinimidyl carbonate is carried out in the aforementionedaprotic solvent or without any solvent. The ratio of N,N′-disuccinimidylcarbonate to be used is not particularly limited but is preferablyequimolar or more, more preferably equimolar to 20 molar to the compound(p). The reaction temperature is preferably 0 to 200° C., morepreferably 20 to 150° C. The reaction time is preferably 10 minutes to48 hours, more preferably 30 minutes to 12 hours. In the reaction, anorganic base such as triethylamine, pyridine, or dimethylaminopyridineor an inorganic base such as sodium carbonate, sodium hydroxide, sodiumhydrogen carbonate, sodium acetate, potassium carbonate, or potassiumhydroxide may be used as a catalyst. The ratio of the catalyst to beused is preferably 0.1 to 50% by weight, more preferably 0.5 to 20% byweight. The compound (c) thus formed may be purified by theaforementioned purification means.

(Process for Producing (b))

Furthermore, the maleimide compound (b) can be obtained by reacting theresulting amine (a) with maleic anhydride in the aforementioned aproticsolvent or without any solvent to obtain an maleimide compound and thensubjecting it to a ring closure reaction using acetic anhydride orsodium acetate as a catalyst. The ratio of maleic anhydride to be usedin the maleamidation reaction is not particularly limited but ispreferably equimolar or more, more preferably equimolar to 5 molar tothe compound (p). The reaction temperature is preferably 0 to 200° C.,more preferably 20 to 120° C. The reaction time is preferably 10 minutesto 48 hours, more preferably 30 minutes to 12 hours. The maleimidecompound formed may be purified by the aforementioned purification meansor may be used as it is in the next ring closure reaction.

The reaction solvent in the subsequent ring closure reaction is notparticularly limited but is preferably aprotic solvent or aceticanhydride. The ratio of sodium acetate to be used is not particularlylimited but is preferably equimolar or more, more preferably equimolarto 50 molar to the maleimide compound. The reaction temperature ispreferably 0 to 200° C., more preferably 20 to 150° C. The reaction timeis preferably 10 minutes to 48 hours, more preferably 30 minutes to 12hours. The compound formed may be purified by the aforementionedpurification means.

The maleimide compound (b) can be also obtained by reacting the compoundof the following formula (b1) with the aforementioned amine (a). Thereaction is carried out in the aforementioned aprotic solvent or withoutany solvent and the compound (b1) is added in an amount of equimolar ormore to the amine (a), followed by reaction. The ratio of (b1) to beused is preferably equimolar or more, more preferably equimolar to 5molar to (a). The reaction temperature is preferably 0 to 200° C., morepreferably 20 to 80° C. The reaction time is preferably 10 minutes to 48hours, more preferably 30 minutes to 12 hours. During the reaction,light shielding may be conducted. The compound formed may be purified bythe aforementioned purification means.

wherein Q represents a hydrocarbon group having 1 to 7 carbon atoms.(Process for Producing (i))

The mercapto compound (i) can be obtained by reacting the compound (p)with methanesulfonyl chloride in the presence of a base to effectmesylation, followed by the reaction with a thiol-forming agent such asthiourea. The reaction solvent in the mesylation reaction is preferablythe above aprotic solvent or without any solvent. The base is preferablyan organic base such as triethylamine, pyridine, or4-dimnethylaminopyridine or an inorganic base such as sodium carbonate,sodium hydroxide, sodium hydrogen carbonate, sodium acetate, potassiumcarbonate, or potassium hydroxide. The ratio of the organic base orinorganic base to be used is not particularly limited but is preferablyequimolar or more to the compound (p). Also, the organic base may beused as a solvent. The ratio of methanesulfonyl chloride to be used isnot particularly limited but is preferably equimolar or more, morepreferably equimolar to 50 molar to the compound (p). The reactiontemperature is preferably 0 to 300° C., more preferably 20 to 150° C.The reaction time is preferably 10 minutes to 48 hours, more preferably30 minutes to 24 hours. The compound formed may be purified by apurification means such as extraction, recrystallization, adsorptiontreatment, reprecipitation, column chromatography, or supercriticalextraction.

The thiol-formation reaction is carried out in a solvent such as water,an alcohol, or acetonitrile or without any solvent. The ratio ofthiourea to be used is equimolar or more, more preferably equimolar to50 molar to the mesylated compound. The reaction temperature ispreferably 0 to 300° C., more preferably 20 to 150° C. The reaction timeis preferably 10 minutes to 48 hours, more preferably 30 minutes to 24hours. After completion of the reaction, the mercapto compound can beobtained by subjecting the resulting thiazolium salt to alkalihydrolysis. The compound formed may be purified by the aforementionedpurification means.

Moreover, the above mercapto compound can be also obtained by reactingthe mesylated compound with the following compound (i1), followed bydecomposition with a primary amine. The reaction of the mesylatedcompound with the compound (i1) is carried out in the aforementionedaprotic solvent or without any solvent. The ratio of the compound (i1)to be used is equimolar or more, more preferably equimolar to 50 molarto the compound (p). The reaction temperature is preferably 0 to 3 00°C., more preferably 20 to 80° C. The reaction time is preferably 10minutes to 48 hours, more preferably 30 minutes to 24 hours. Thesubsequent alkali decomposition with a primary amine is carried out inthe aforementioned aprotic solvent or without any solvent. The primaryamine to be used is not particularly limited but preferably includesammonia, methylamine, ethylamine, propylamine, butylamine, pentylamine,hexylamine, cyclohexylamine, ethanolamine, propanolamine, butanolamine,and the like. Naturally, the primary amine may be used as a solvent. Thecompound formed may be purified by the aforementioned purificationmeans.

(Process for Producing (e))

A haloacetyl compound (e) can be, for example, synthesized by thefollowing process. It can be obtained by reacting the compound (p) orthe compound (a) with an organic base such as triethylamine, pyridine,or dimethylaminopyridine or an inorganic base such as sodium carbonate,sodium hydroxide, sodium hydrogen carbonate, sodium acetate, potassiumcarbonate, or potassium hydroxide and a compound represented by thefollowing general formula (e1) in an aprotic solvent such as toluene,benzene, xylene, acetonitrile, ethyl acetate, diethyl ether, t-butylmethyl ether, tetrahydrofuiran, chloroform, methylene dichloride,dimethyl sulfoxide, dimethylformamide, or dimethylacetamide, or withoutany solvent. The ratio of the organic base or inorganic base to be usedis not particularly limited but is preferably equimolar or more to thecompound (p) or (a). Furthermore, an organic base may be used as asolvent. When the compound (a) is used, the above organic base orinorganic base may not be used. W¹ in (e1) is a halogen atom selectedfrom Cl, Br, and I, and is preferably I. Moreover, W¹s may be the sameor different from each other. The ratio of the compounds represented bythe general formula (e1) to be used is not particularly limited but ispreferably equimolar or more, more preferably equimolar to 50 molar tothe compound (p) or (a). The reaction temperature is preferably 0 to300° C., more preferably 20 to 150° C. The reaction time is preferably10 minutes to 48 hours, more preferably 30 minutes to 24 hours. Thecompound formed may be purified by a purification means such asextraction, recrystallization, adsorption treatment, reprecipitation,column chromatography, or supercritical extraction.

(Process for Producing (f))

The compound (f) can be, for example, prepared by the following process.The hydrazine derivative of (f) can be obtained by condensing thecompound (h) with the following compound (f1) in the presence of acondensing agent such as DCC, EDC, or BOP[(benzotriazolyloxy)tris(dimethylamino)phosphonium hexafluorophosphate].The reaction of the compound (h) with (f1) is carried out in the aboveaprotic solvent or without any solvent. The ratio of (f1) to be used isnot particularly limited but is preferably equimolar or more, morepreferably equimolar to 5 molar to the compound (h). The condensingagent is not particularly limited but is preferably DCC, EDC, or BOP.The ratio of the condensing agent to be used is not particularly limitedbut is preferably equimolar or more, more preferably equimolar to 5molar to the compound (h). The reaction temperature is preferably 0 to100° C., more preferably 20 to 80° C. The reaction time is preferably 10minutes to 48 hours, more preferably 30 minutes to 12 hours. Thecompound formed may be purified by the aforementioned purificationmeans.NH₂NH—Boc   (f1)wherein the Boc group means a t-butoxycarbonyl group.

Subsequent deprotection of the Boc group can be carried out by a knownmethod. The compound (t) synthesized may be purified by theaforementioned purification means.

(Process for Producing (g))

The compound (g) can be, for example, prepared by the following process.The hydroxylamine derivative of (g) can be obtained by condensing thecompound (p) or (a) with the following compound (g1) in the presence ofa condensing agent such as DCC, EDC, or BOP[(benzotriazolyloxy)tris(dimethylamino)phosphonium hexafluorophosphate].The reaction of the compound (p) or (a) with (g1) is carried out in theabove aprotic solvent or without any solvent. The ratio of (g1) to beused is not particularly limited but is preferably equimolar or more,more preferably equimolar to 5 molar to the compound (p) or (a). Thecondensing agent is not particularly limited but is preferably DCC, EDC,or BOP. The ratio of the condensing agent to be used is not particularlylimited but is preferably equimolar or more, more preferably equimolarto 5 molar to the compound (p) or (a). The reaction temperature ispreferably 0 to 100° C., more preferably 20 to 80° C. The reaction timeis preferably 10 minutes to 48 hours, more preferably 30 minutes to 12hours. The compound formed may be purified by the aforementionedpurification means.

Subsequent deprotection of the Boc group can be carried out by a knownmethod. The compound (g) synthesized may be purified by theaforementioned purification means.

(Process for Producing (j))

The aldehyde compound (j) can be obtained by reacting the mesylatedproduct of the compound (p) with an acetal compound (j1) to obtain anacetal compound and then subjecting it to hydrolysis under an acidiccondition. The production of the mesylated compound is as shown in theproduction process of (i). The acetalization reaction can be achieved bythe reacting with an equimolar or more, preferably an equimolar to 50molar of a (j1) to the mesylated compound in the aforementioned aproticsolvent or without any solvent. (j1) can be prepared from thecorresponding alcohol using sodium, potassium, sodium hydride, potassiumhydride, sodium methoxide, potassium t-butoxide, or the like. Thereaction temperature is preferably 0 to 300° C., more preferably 20 to150° C. The reaction time is preferably 10 minutes to 48 hours, morepreferably 30 minutes to 24 hours.

In the case of using (j2), an acetal compound can be obtained byconverting the hydroxyl group of the compound (p) into an alcoholate bythe aforementioned process and then reacting it with an equimolar ormore, preferably an equimolar to 100 molar of (j2) in the aforementionedaprotic solvent or without any solvent. The reaction temperature ispreferably 0 to 300° C., more preferably 20 to 150° C. The reaction timeis preferably 10 minutes to 48 hours, more preferably 30 minutes to 24hours.

In the case of using (j3), an acetal compound can be obtained byreacting (j3) with (c), (d), or (h). The production of (c), (d), or (h)is as mentioned above. In the reaction with (j3), the solvent is notparticularly limited but the reaction is preferably carried out in theaforementioned aprotic solvent. The charging ratio of (j3) is preferablyequimolar or more, more preferably equimolar to 10 molar to (c), (d), or(h). The reaction temperature is preferably −30 to 200° C., morepreferably 0 to 150° C. The reaction time is preferably 10 minutes to 48hours, more preferably 30 minutes to 24 hours. In the case of using (h),a condensing agent such as DCC or EDC may be optionally used. Anyacetalization reaction may be carried out under light shielding. Theacetal compound thus obtained may be purified by the aforementionedpurification means or may be used as it is in the nextaldehyde-formation reaction.

The aldehyde compound can be produced by dissolving the acetal compoundto form a 0.1 to 50% aqueous solution and hydrolyzing it in an aqueoussolution which is adjusted to pH 1 to 4 with an acid such as aceticacid, phosphoric acid, sulfuric acid, or hydrochloric acid. The reactiontemperature is preferably −20 to 100° C., more preferably 0 to 80° C.The reaction time is preferably 10 minutes to 24 hours, more preferably30 minutes to 10 hours. The reaction may be carried out under lightshielding. The compound formed may be purified by the aforementionedpurification means.

wherein R² and R³ are each a hydrocarbon group having 1 to 3 carbonatoms and may be the same or different from each other, and they maytogether form a ring; M is sodium or potassium; W² is a halogen atomselected from Cl, Br, and I; and t is an integer of 1 to 5.

In the polyalkylene glycol derivative represented by the formula (1)thus obtained, an amount of ionic functional group-containing impuritiesother than the target compound, i.e., reactive impurities is 1% or less,more preferably 0.5% or less, futher preferably 0.2% or less in achromatogram obtained by analysis by liquid chromatography using anion-exchange column.

FIG. 3 is a model chart of a chromatogram obtained by liquidchromatography using an ion-exchange column.

In the liquid chromatography using an ion-exchange column, a moleculemay interact with the column depending on the charge of the molecule anda molecule having a larger charge elutes more slowly. Namely, in FIG. 3,a first eluting peak A shows a compound having a non-ionic functionalgroup, a peak B shows a target compound, a peak C shows a reactiveimpurity having a molecular weight 0.5 time that of the target compound,and a peak D shows a bifunctional reactive impurity having the samemolecular weight as that of the target compound. Namely, in thechromatogram of FIG. 3, ionic functional group-containing impuritiesother than the target compound are shown by the peaks C and D.

Accordingly, the amount (%) of the ionic functional group-containingimpurities other than the target compound is calculated by the followingexpression.(Area of peak C+Area of peak D)/(Total peak area)×100

In the case that an elution position is unknown, the compound can besuitably identified using an authentic sample.

In the case that the target compound has no ionic functional group, thecompound is measured after it is reacted with a labeling agent having anionic functional group. For example, in the case of (b) in the group I,it can be measured after labeled by the reaction with mercaptopropionicacid to convert a functional group into a carboxyl group. Moreover, inthe case of (d) in the group (I), it can be measured after labeled bythe reaction with glycine to convert a functional group into a carboxylgroup.

When the amount of the reactive impurities is larger than 1%, a reactionproduct with a bio-related substance becomes heterogeneous, whichinfluences performance of the resulting modified bio-related substance.

In the invention, the liquid chromatography using an ion-exchange columnis measured under the following conditions:

-   (In the case that an amine compound in the group I(a) is measured)-   HPLC apparatus: Alliance2695 (Nihon Waters K.K.); column: TSK-gel    SP-5PW (manufactured by Tosoh Corporation); eluent: sodium phosphate    buffer solution (pH 6.5); column temperature: 40° C.; flow rate: 0.5    ml/min; detector: differential refractometer detector (RI) (Nihon    Waters K.K.); sample concentration: 5 mg/ml; injection amount: 20    μl.-   (In the case that a carboxyl compound in the group I(h) is measured)-   HPLC apparatus: Alliance2695 (Nihon Waters K.K.); column: ES-502N    (Asahipak); eluent: ammonium formate buffer solution (pH 8.0);    column temperature: 30° C.; flow rate: 1.0 ml/min; detector:    differential refractometer detector (RI) (Nihon Waters K.K.); sample    concentration: 10 mg/ml; injection amount: 20 μl.

In the (b) in the group I of the polyalkylene glycol derivative of theformula (1) of the invention, when a nuclear magnetic resonance spectrumis obtained as a deuterated methanol solution, an integral value M1detected at around 6.84 ppm, which is originated from the maleimidegroup derived from the hydroxyl group at the 1-position directly bondedto the glycerin skeleton in the case that n is 0, and an integral valueM2 detected at around 6.84 ppm, which is originated from the maleimidegroup derived from the hydroxyl group of the polyalkylene glycol chainsatisfy a relationship:M2/(M1+M2)×100≦2.More preferably, they satisfy a relationship:M2/(M1+M2)×100≦1.Further preferably, they satisfy a relationship:M2/(M1+M2)×100≦5.

The following will illustrate the calculation method of M1 and M2.Twenty miligrams of (b) in the group I is dissolved in deuteratedmethanol and a ¹H nuclear magnetic resonance spectrum is measured. Anintegral value detected at around 6.84 ppm is defined as M1, which isoriginated from the maleimide group derived from the hydroxyl group atthe 1-position directly bonded to the glycerin skeleton in the case thatn is 0, a TMS standard peak being defined as 0 ppm. Moreover, anintegral value detected at around 6.84 ppm, which is originated from themaleimide group derived from the oxyalkylene chain end or the thepolyalkylene glycol chain formed by decomposition is defined as M2.

In the case that M2/(M1+M2)×100, which is derived from M1 and M2 thusdetermined, is larger than 2, since impurity having a 0.5 time molecularweight, bifunctional and trifunctional impurities having the samemolecular weight, low-molecular-weight impurities, and the like arecontained-in large amounts, purity of the modified bio-related substancetends to decrease.

According to the invention, there can be obtained a novel branchedpolyalkylene glycol derivative having a reactive group capable of beingcombined with a bio-related substance at the primary carbon at the1-position of the glycerin skeleton and having polyalkylene glycolchains at 2- and 3-positions. Moreover, a bio-related substance modifiedwith the branched polyalkylene glycol derivative can be obtained. Thebio-related substance is formed by ether bonds only except for thelinker part with the poly(alkylene glycol)oxy group, so that a highstability can be expected with no decomposition into a single chain.Therefore, by modifying a bio-related substance with the branchedpolyalkylene glycol, a bio-related substance exhibiting an improvedbehavior in a body can be provided.

EXAMPLES

The following will describe the present invention more specifically withreference to Examples. In this regard, ¹H-NMR, GPC, and liquidchromatography were employed for analyzing and identifying the compoundsin Examples.

<Method for ¹H-NMR Analysis>

At ¹H-NMR analysis, JNM-ECP400 manufactured by Nippon Denshi Datum K.Kwas employed. The integral values in NMR data are theoretical values.

(Method for GPC Analysis):

At GPC analysis, LC10AVP was employed as a GPC system and measurementwas carried out under the following conditions: column: PLgel MIXED-D(manufactured by Polymer Raboratory) two columns; developing solvent:DMF (containing 10 mM lithium bromide); flow rate: 0.7 ml/min; columntemperature: 65° C.; detector: RI; sample amount: 1 mg/mL, 100 μl.

At analysis of an aldehyde compound, measurement was carried out underthe following conditions:

-   system: Alliance2695 (Nihon Waters K.K.); developing solvent: 100 mM    sodium acetate buffer (pH=5.2, containing 0.02% NaN₃); flow rate:    0.5 ml/min; column:-   Ultahydrogel500+Ultlahydrogel250 2 columns; column temperature: 30°    C.; detector: RI; sample amount: 5 mg/mL, 20 μl.

In GPC data, analysis values at main peaks which are obtained by cuttingelution curves perpendicular to base lines at inflection points toremove high-molecular-weight impurities and low-molecular-weightimpurities and analysis values over whole peaks from start points ofelution to end points of elution were included.

Mn represents a number average molecular weight, Mw represents a weightaverage molecular weight, and MP represents a peak top molecular weight.

(Analytic Method for Liquid Chromatography):

The liquid chromatography using an ion-exchange column is measured underthe following conditions:

-   (In the case that an amine compound is measured)-   HPLC apparatus: Alliance2695 (Nihon Waters K.K.); column: TSK-gel    SP-5PW (manufactured by Tosoh Corporation); eluent: sodium phosphate    buffer (pH 6.5); column temperature: 40° C.; flow rate: 0.5 ml/min;    detector: differential refractometer detector (RI) (Nihon Waters    K.K.); sample concentration: 5 mg/ml; injection amount: 20 μl.-   (In the case that a carboxyl compound is measured)-   HPLC apparatus: Alliance2695 (Nihon Waters K.K.); column: ES-502N    (Asahipak); eluent: ammonium formate buffer (pH 8.0); column    temperature: 30° C.; flow rate: 1.0 ml/min; detector: differential    refractometer detector (RI) (Nihon Waters K.K.); sample    concentration: 10 mg/ml; injection amount: 20 μl.

Example 1 Synthesis of compound (p) (synthesis of R=methyl group,A²O=oxyethylene group, m=223, and m=475) Example 1-1

To a 1000 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, and a stirrer were added 132.2 g (1.0 mol) of2,2-dimethyl-1,3-dioxolane-4-methanol, 202.5 g (1.05 mol) of a 28%methanol solution of sodium methoxide, and 600 ml of toluene. Withintroducing nitrogen thereinto, the toluene was refluxed under reducedpressure for 1 hour to remove the methanol by distillation. Withmaintaining the solution at 80° C., 126.6 g (1.0 mol) of benzyl chloridewas added dropwise over a period of 2 hours using a dropping funnel,followed by further 2 hours of reaction. After completion of thereaction, the temperature was lowered to 60° C. and 10 g of KYOWAAD 600was added thereto, followed by 1 hour of stirring. The reaction liquidwas filtrated, the solvent was removed, and the residue was purified bydistillation (b.p. 93-95° C./266 Pa) to obtain4-(benzyloxymethyl)-2,2-dimethyl-1,3-dioxolane.

¹H-NMR (CDCl₃, internal standard: TMS) δ(ppm): 1.36, 1.42 (3H, 3H, s,C(CH ³ )₂), 3.45-3.57 (2H, m, CH ² O—C(CH₃)₂), 3.73-3.76 (1H, m,CHO—C(CH₃)₂), 4.03-4.07, 4.28-4.32 (2H, m, CH ² O—CH₂Ph), 4.57 (2H, q,—CH ² Ph), 7.15-7.40 (5H, m, —CH₂ Ph)(Ph represents a phenyl group)

Example 1-2

To 222 g (1.0 mol) of 4-(benzyloxymethyl)-2,2-dimethyl-1,3-dioxolane wasadded 350 g of distilled water, and the whole was adjusted to pH 2 withphosphoric acid. With introducing nitrogen thereinto, the solution washeated to 70 ° C. After 2 hours of reaction, the solution was adjustedto pH 7.0 with sodium hydroxide. After 1 L of chloroform was addedthereto and extraction was effected, the resulting chloroform layer wasdried over magnesium sulfate and concentrated and the resulting saltswere removed by filtrating the condensate to obtain a compound (pa)which was 3-benzyloxy-1,2-propanediol.

¹H-NMR (CDCl₃, internal standard: TMS) δ(pm): 3.50-3.71 (4H, m, CH ² OH,CH ² O—CH₂Ph), 3.86-3.91 (1H, m, CHOH), 4.54 (2H, m, —CH ² Ph),7.27-7.38 (5H, m, —CH₂ Ph).

Example 1-3

To a 300 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, and a pressure-reducing line wereadded 27.3 g (0.15 mol) of 3-benzyloxy-1,2-propanediol, 200 g of drytoluene, and 0.76 g of sodium. With introducing nitrogen thereinto, thewhole was heated to 35° C. to dissolve sodium. The solution was chargedinto a 5 L autoclave thoroughly dried beforehand and the atmosphere wasreplaced by nitrogen, followed by heating to 100 ° C. Then, 3100 g ofethylene oxide was added thereto at 100 to 150 ° C. under a pressure of1 MPa or lower, followed by continuation of the reaction for another 1.5hours. Unreacted ethylene oxide gas and toluene were removed bydistillation under reduced pressure, and then the whole was cooled to70° C. After 2.0 kg of the reaction liquid was taken out of theautoclave, the liquid was adjusted to pH 7.5 with 85% aqueous phosphoricacid solution to obtain the following compound (p1).

¹H-NMR (CDCl₃, internal standard: TMS) δ(ppm): 3.40-3.80 (1789H, m, —CH² O(CH ² CH ² O)_(m)H, CHO(CH ² CH ² O)_(m)H, CH ² OCH₂Ph), 4.54 (2H, s,—CH ² Ph), 7.27-7.38 (5H, m, —CH₂ Ph).

Example 1-4

Into a 2 L round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were added 200 g (10 mmol) of the above compound (p1) and 1000 g oftoluene, and the whole was heated under reflux to effect azeotropicremoval of 200 g of toluene and water. After cooling to roomtemperature, 4.05 g (40 mmol) of triethylamine was added thereto and,after heating to 40° C., 3.44 g (30 mmol) of methanesulfonyl chloridewas added dropwise thereto, followed by 3 hours of reaction at 40° C.After the reaction was finished, 19.28 g (50 mmol) of 28% methanolsolution of sodium methoxide was added thereto, followed by 3 hours ofreaction at 40° C. The pressure was reduced with maintaining thereaction liquid at 40° C. and about 200 g of a mixed liquid ofmethanol/toluene was removed by evaporation, and then salts were removedby filtration. Then, 500 g of toluene was added to the filtrate and theresulting filtrate was transferred into a 2 L round-bottom flask fittedwith a thermometer, a nitrogen-introducing tube, a stirrer, a Dean-starktube, and a condenser tube, followed by heating under reflux to effectazeotropic removal of 200 g of toluene and water. After cooling to roomtemperature, 4.05 g (40 mmol) of triethylamine was added thereto and,after heating to 40° C., 3.44 g (30 mmol) of methanesulfonyl chloridewas again added dropwise thereto, followed by 3 hours of reaction at 40°C. After completion of the reaction, 19.28 g (100 mmol) of 28% methanolsolution of sodium methoxide was added thereto, followed by 3 hours ofreaction at 40° C. The pressure was reduced with maintaining thereaction liquid at 40° C. and about 200 g of a mixed liquid ofmethanol/toluene was removed by distillation, followed by removal ofsalts by filtration. The filtrate was heated to 50° C. and 200 g of 25%aqueous sodium chloride solution was added thereto. After stirring, thewhole was left on standing to separate into layers and the lower waterlayer was removed. This operation of washing with water was repeatedtwice. The upper toluene layer was dried over magnesium sulfate and thenfiltrated and 1 L of ethyl acetate was added to the filtrate. Hexane wasadded thereto until crystals were precipitated. The crystals werecollected by filtration and dried to obtain the following compound (p2).

¹H-NMR (CDCl₃, internal standard: TMS) δ(ppm): 3.38 (6H, s, —CH₃),3.40-3.80 (1789H, m, —CH ² O(CH ² CH ² O)_(m)CH₃, CHO(CH ² CH ²O)_(m)CH₃, CH ² OCH₂Ph), 4.54 (2H, s, —CH ² Ph), 7.27-7.38 (5H, m, —CH₂Ph).

Example 1-5

Water removal of palladium carbon was carried out by adding 120 g of 5%palladium carbon (50% hydrous product, manufactured by N. E. M. Cat.)into a pressure filter and replacing the solvent by 500 ml of drymethanol four times with replacement of the atmosphere by nitrogen. Intoa 2 L round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, and a condenser tube were added100 g of the above compound (p2), and the whole amount of thepalladium-carbon subjected to solvent replacement. After the replacementby nitrogen, 1200 ml of dry methanol and 500 ml of cyclohexene wereadded thereto and the whole was heated to 30° C. to be allowed to reactfor 3.5 hours. The reaction liquid was filtrated and the filtrate wasconcentrated. Then, 1 L of ethyl acetate was added and hexane was addedthereto until crystals were precipitated. The resulting crystals werecollected by filtration and dried to obtain the following compound (p3).

¹H-NMR (CDCl₃, internal standard: TMS) δ(ppm): 3.38 (6H, s, —CH ³ ),3.40-3.80 (1789H, m, —CH ² O(CH ² CH ² O)_(m)CH₃, CHO(CH ² CH ²O)_(m)CH₃, CH ² OH).

Example 1-6

In Example 1-3, the autoclave where about 1 kg of the reaction liquidremained was replaced with nitrogen and was heated to 120° C. Then, 1190g of ethylene oxide was added thereto at 100 to 150° C. under a pressureof 1 MPa or lower, followed by continuation of the reaction for another4 hours. After completion of the reaction, unreacted ethylene oxide gaswas removed with introducing nitrogen gas into a reaction liquid andthen the whole was cooled to 80° C. and the liquid was adjusted to pH7.5 with 85% aqueous phosphoric acid solution to obtain the followingcompound (p4).

¹H-NMR (CDCl₃, internal standard: TMS) δ(ppm): 3.40-3.80 (3805H, m, —CH² O(CH ² CH ² O)_(m)H, CHO(CH ² CH ² O)_(m)H, CH ² OCH₂Ph), 4.54 (2H, s,—CH ² Ph), 7.27-7.38 (5H, m, —CH₂ Ph)

Example 1-7

Into a 2 L round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were added 252 g (6 mmol) of the above compound (p4) and 1000 g oftoluene, and the whole was heated under reflux to effect azeotropicremoval of 200 g of toluene and water. After cooling to roomtemperature, 2.43 g (24 mmol) of triethylamine was added thereto and,after heating to 40° C., 2.06 g (18 mmol) of methanesulfonyl chloridewas added dropwise thereto, followed by 3 hours of reaction at 40° C.After completion of the reaction, 6.94 g (36 mmol) of 28% methanolsolution of sodium methoxide was added thereto, followed by 3 hours ofreaction at 40° C. The pressure was reduced with maintaining thereaction liquid at 40° C. and about 200 g of a mixed liquid ofmethanol/toluene was removed by distillation, followed by removal ofsalts by filtration. Then, 500 g of toluene was added to the resultingfiltrate and the resulting mixture was transferred into a 2 Lround-bottom flask fitted with a thermometer, a nitrogen-introducingtube, a stirrer, a Dean-stark tube, and a condenser tube, followed byheating under reflux to effect azeotropic removal of 200 g of tolueneand water. After cooling to room temperature, 2.43 g (24 mmol) oftriethylamine was added thereto and, after heating to 40° C., 2.06 g (18mmol) of methanesulfonyl chloride was again added dropwise thereto,followed by 3 hours of reaction at 40° C. After completion of thereaction 6.94 g (36 mmol) of 28% methanol solution of sodium methoxidewas added thereto, followed by 3 hours of reaction at 40° C. Thepressure was reduced with maintaining the reaction liquid at 40° C. andabout 200 g of a mixed liquid of methanol/toluene was removed bydistillation, followed by removal of salts by filtration. The filtratewas heated to 50° C. and 200 g of 25% aqueous sodium chloride solutionwas added thereto. After stirring, the whole was left on standing toseparate into layers and the lower water layer was removed. Thisoperation of washing with water was repeated twice. The upper toluenelayer was dried over magnesium sulfate and then filtrated and 1 L ofethyl acetate was added to the filtrate. Hexane was added thereto untilcrystals were precipitated. The crystals were collected by filtrationand dried to obtain the following compound (p5).

¹H-NMR (CDCl₃, internal standard: TMS) δ(ppm): 3.38 (6H, s, —CH ³ ),3.40-3.80 (3805H, m, —CH ² O(CH ² CH ² O)_(m)CH₃, CHO(CH ² CH ²O)_(m)CH₃, CH ² OCH₂Ph), 4.54 (2H, s, —CH ² Ph), 7.27-7.38 (5H, m, —CH₂Ph).

Example 1-8

Water removal of palladium carbon was carried out by adding 200 g of 5%palladium carbon (50% hydrous product, manufactured by N. E. M. Cat.)into a pressure filter and replacing the solvent by 500 ml of drymethanol four times with replacement of the atmosphere by nitrogen. Intoa 2 L round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, and a condenser tube were added100 g of the above compound (p5) and the whole amount of thepalladium-carbon subjected to solvent replacement. After the replacementby nitrogen, 1200 ml of dry methanol and 500 ml of cyclohexene wereadded thereto and the whole was heated to 30° C. to be allowed to reactfor 3.5 hours. The reaction liquid was filtrated and the filtrate wasconcentrated. Then, 1 L of ethyl acetate was added and hexane was addedthereto until crystals were precipitated. The resulting crystals werecollected by filtration and dried to obtain the following compound (p6).

¹H-NMR (CDCl₃, internal standard: TMS) δ(ppm): 3.38 (6H, s, —CH ³ ),3.40-3.80 (38051H, m, —CH ² O(CH ² CH ² O)_(m)CH₃, CHO(CH ² CH ²O)_(m)CH₃, CH ² OH).

Example 2 Synthesis of amino compound (group I(a)) (R=methyl group,A²O=oxyethylene group, m=475) Example 2-1

Into a 500 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, and a condenser tube was added52.4.g of the above compound (p6). Then, 52.4 g of ion-exchanged waterand 3.3 g of 50% aqueous potassium hydroxide solution were addedthereto, and the whole was heated to 40° C. to dissolve them withnitrogen bubbling. After dissolution, the solution was cooled to 10° C.or lower and 146 g of acrylonitrile was added dropwise over a period of2 hours with maintaining a temperature of 5 to 10° C. After the dropwiseaddition, the reaction was continued for another 2 hours. The nitrogenbubbling was continued during the reaction. Then, 19.5 g of 8.5% aqueousphosphoric acid solution was added dropwise, followed by neutralizationof the reaction liquid. Subsequently, 90 g of ion-exchanged water, 116 gof ethyl acetate, and 12 g of hexane were added to the reaction liquidand, after 10 minutes of stirring, the whole was left on standing for 20minutes. The upper ethyl acetate layer was removed with suction by meansof a peristaltic pump. Subsequently, 116 g of ethyl acetate was addedthereto and, after stirred for 10 minutes, the whole was left onstanding for 20 minutes. The upper ethyl acetate layer was removed withsuction by means of a peristaltic pump. The extraction with ethylacetate was repeated eight times. After completion of the extraction, 31g of sodium chloride was added to the aqueous layer and dissolvedtherein, and then the solution was extracted with 300 g of chloroform.The resulting chloroform layer was dried over magnesium sulfate,filtrated, and then concentrated. Thereafter, 460 g of ethyl acetate wasadded to the concentrate, which was dissolved therein. Then, hexane wasadded thereto until crystals were precipitated. The crystals werecollected by filtration and dispersed in 230 g of hexane. The crystalswere again collected by filtration and dried to obtain the followingnitrile compound (p7).

¹H-NMR (CDCl₃, internal standard: TMS) δ(ppm): 2.59-2.66 (2H, m, —CH ²CH₂CN), 3.38 (6H, s, —CH ³ ), 3.40-3.80 (3807H, m, —CH ² O(CH ² CH ²O)_(m)CH₃, CHO(CH ² CH ² O)_(m)CH₃, CH ² OCH₂ CH ² CN).

Example 2-2

To a 1 L autoclave were added 50 g of the nitrile compound of theformula (p7), 400 g of toluene, 4.51 g of nickel (manufactured by N. E.M. Cat., 5136p), 0.89 g of 28% aqueous ammonia, and 0.05 g of BHT. Afterreplacement of the atmosphere with nitrogen, the autoclave was pressuredwith nitrogen until the inner pressure reached 0.3 MPa. Subsequently,the autoclave was pressurized with hydrogen until the inner pressurereached 3.5 MPa. Then, the autoclave was heated to 130° C. andpressurized with hydrogen until the inner pressure reached 4.0 MPa,followed by 3 hours of reaction at 130° C. After completion of thereaction, the reaction liquid was cooled to 70° C., and purge withnitrogen was repeated three times. The whole amount of the reactionliquid was taken out and, after removal of water by dispersing 80 g ofmagnesium sulfate, the mixture was filtrated to remove the nickelcatalyst. After the filtrate was concentrated to about 300 ml and cooledto room temperature, hexane was added until crystals were precipitated.The crystals were collected by filtration and dried to obtain thefollowing amine compound (p8).

¹H-NMR (D₂O, internal standard: H₂O=4.7 ppm) δ(ppm): 1.82-1.90 (2H, m,—CH₂CH₂ CH ² NH₂), 2.90-2.97 (2H, m, —CH₂ CH ² CH₂NH₂), 3.38 (6H, s, —CH³ ), 3.40-3.80 (3807H, m, —CH ² O(CH ² CH ² O)_(m)CH₃, CHO(CH ² CH ²O)_(m)CH₃, CH ² OCH ² CH₂CH₂NH₂).

<main peak> number average molecular weight (Mn): 41514, weight averagemolecular weight (Mw): 42234, polydispersity (MwMn): 1.017, peak topmolecular weight (Mp): 41826;

<whole peak> number average molecular weight (Mn): 40259, weight averagemolecular weight (Mw): 41826, polydispersity (Mw/Mn): 1.039, peak topmolecular weight (Mp): 42525.

Low-molecular-weight impurities: 2.82%

Example 2-3

When the compound of the formula (p8) was analyzed on a liquidchromatography using an ion-exchange column (concentration of sodiumphosphate buffer solution: 0.2 mM), any ionic functionalgroup-containing impurity other than the target compound was notdetected.

Example 3-1 Synthesis of maleimide compound (group I(b)) (R=methylgroup, A²O=oxyethylene group, m=475)

Into a 300 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, and a condenser tube were added 20g (0.44 mmol) of the compound (p8), 16 g of acetonitrile, and 104 ml oftoluene, and the whole was heated at 40° C. to dissolve them. Aftercooling to room temperature, 0.24 g (2.37 mmol) of N-methylmorpholineand 190 mg (0.71 mmol) of N-succinimidyl 3-maleimidopropionate wereadded thereto under light shielding, followed by 3.5 hours of reaction.After filtration of the reaction liquid, 360 g of ethyl acetate wasadded and 264 g of hexane was added thereto to precipitate crystals. Thecrystals were collected by filtration and 16 g of acetonitrile and 365 gof ethyl acetate were added. After dissolution under heating, 268 g ofhexane was added to precipitate crystals. The crystallization operationwas again performed. Then, the crystals were collected by filtration anddried to obtain the following compound (p9). The series of thecrystallization operations were carried out under light shielding.

¹H-NMR (CDCl₃, internal standard: TMS) δ(pm): 1.70-1.78 (2H, m, —CH₂ CH² CH₂N), 2.45-2.53 (2H, m, —NHCOCH ² CH₂N), 3.38 (6H, s, —CH₃),3.40-3.80 (3811H, m, —CH ² O(CH ² CH ² O)_(m)CH₃, CHO(CH ² CH ²O)_(m)CH₃, CH ² OCH ² CH₂ CH ² NHCOCH₂ CH ² ), 6.44 (1H, m, NHCO), 6.71(2H, s, —CH═CH—).

<main peak> number average molecular weight (Mn): 40886, weight averagemolecular weight (Mw): 41597, polydispersity (Mw/Mn): 1.017, peak topmolecular weight (Mp): 41928;

<whole peak> number average molecular weight (Mn): 39853, weight averagemolecular weight (Mw): 41562, polydispersity (Mw/Mn): 1.043, peak topmolecular weight (Mp): 41928.

Low-molecular-weight impurities: 2.84%

Example 3-2

Twenty miligrams of the compound of the formula (p9) was dissolved in 2ml of aqueous mercaptopropionic acid solution (1 mg/ml). The solutionwas stirred at room temperature for 3 hours under light shielding. Whenthe solution was analyzed on a liquid chromatography using anion-exchange column (concentration of ammonium formate buffer solution:0.3 mM), any ionic functional group-containing impurity other than thetarget compound was not detected.

Example 3-3

Twenty miligrams of the compound (p9) was dissolved in deuteratedmethanol. The solution was measured on ¹H-NMR (number of integrationtimes: 256) and M2/(M1+M2)×100 was calculated. M1 M2 M2/(M1 + M2) × 1002 0.0170 0.84

Example 4 Synthesis of succinimide ester compound (group I(c)) (R=methylgroup, A²O=oxyethylene group, m=475)

Into a 500 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, and a condenser tube were charged55 g (1.31 mmol) of the compound of the formula (p8), 238 g of drytoluene, 0.055 g of BHT, and 0.55 g of sodium acetate, and the whole washeated to dissolved them. The reaction liquid was heated to 55° C. and1.196 g of glutaric anhydride was added thereto, followed by 5 hours ofreaction at 55° C. Thereafter, 2.41 g of N-hydroxysuccininide was addedthereto at 55° C. After 1 hour of stirring, the reaction liquid wascooled to 40° C. After cooling, 4.33 g of DCC was added thereto,followed by 4 hours of reaction. After completion of the reaction, thereaction liquid was filtered to remove DCU. Then, 385 g of hexane wasadded to the filtrate to precipitate crystals and the crystals werecollected by filtration. The collected crystals were dissolved underheating with adding 44 g of acetonitrile and 396 g of ethyl acetate.Then, 286 g of hexane was added thereto to precipitate crystals and thecrystals were collected by filtration. The crystallization operation wasrepeated five times and the resulting crystals were dried to obtain thecompound of the following (p10).

¹H-NMR (CDCl₃, internal standard: TMS) δ(ppm): 1.76 (2H, m, —OCH₂ CH ²CH₂NHCOCH₂CH₂CH₂—), 2.10 (2H, m, —NHCOCH₂ CH ² CH₂COON—), 2.32 (2H, t,—NHCOCH ² CH₂CH₂COON—), 2.69 (2H, t, —NHCOCH₂ CH ² COON—), 2.86 (4H, s,succinimide), 3.38 (6H, s, —CH ³ ), 3.40-3.80 (4043H, m, —CH ² O(CH ²OCH ² CH ² O)_(m)CH₃, CHO(CH ² CH ² O)_(m)CH₃, —CH ² —OCH ² CH₂ CH ²NHCOCH₂CH₂CH₂—).

<main peak> number average molecular weight (Mn): 41867, weight averagemolecular weight (Mw): 42608, polydispersity (Mw/Mn): 1.018, peak topmolecular weight (Mp): 42999;

<whole peak> number average molecular weight (Mn): 39999, weight averagemolecular weight (Mw): 41913, polydispersity (Mw/Mn): 1.048, peak topmolecular weight (Mp): 42999.

Low-molecular-weight impurities: 3.75%

Example 5-1 Synthesis of p-nitrophenyl carbonate compound (group I(d))(R=methyl group, A²O=oxyethylene group, m=475)

Into a 500 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were charged 130 g of the compound of the formula (p6) and 420 g oftoluene, and the whole was heated under reflux to effect azeotropicremoval of water. The reaction liquid was cooled to 80° C. and 3.13 g oftriethylamine and 4.99 g of p-nitrophenyl chloroformate were addedthereto, followed by 5 hours of reaction at 80° C. After the reactionliquid was filtered, 1560 g of ethyl acetate was added and the whole wascooled to 30° C. Thereafter, 780 g of hexane was added to precipitatecrystals and the crystals were collected by filtration. The collectedcrystals were dissolved under heating with adding 1300 g of ethylacetate. Then, 520 g of hexane was added thereto to precipitate crystalsand the crystals were collected by filtration. The crystallizationoperation was repeated five times and the resulting crystals were driedto obtain the compound of the following (p11).

¹H-NMR (CDCl₃, internal standard: TMS) δ(pm): 3.38 (6H, s, —CH ³ ),3.40-3.80 (4043H, m, —CH ² O(CH ² CH ² O)_(m)CH₃, CHO(CH ² CH ²O)_(m)CH₃), 4.34-4.48 (2, m, —CH ² OCOO—Ph—NO₂), 7.40, 8.28 (2H, 2H, d,d, —CH₂OCOO—Ph—NO₂).

<main peak> number average molecular weight (Mn): 41686, weight averagemolecular weight (Mw): 42429, polydispersity (Mw/Mn): 1.018, peak topmolecular weight (Mp): 42813;

<whole peak> number average molecular weight (Mn): 40190, weight averagemolecular weight (Mw): 41828, polydispersity (Mw/Mn): 1.041, peak topmolecular weight (Mp): 42813.

Low-molecular-weight impurities: 2.99%

Example 5-2

To 50 mg of the compound of the formula (p11) were added 10 mg ofglycine and 1 ml of 0.1M phosphate buffer solution (pH 8.5), followed bydissolving them. The whole was stirred at room temperature for 20 hours.To a gel filtration column equilibrated with 0.3 mM ammonium formatebuffer solution was added 2 ml of a solution of the reaction liquiddiluted five times with the buffer solution. Furthermore, the buffersolution was added thereto and a high-molecular-weight fraction whichwas first eluted was collected into a vial for HPLC measurement. Then,when the fraction was analyzed by liquid chromatography (concentrationof ammonium formate buffer solution: 0.3 mM), any ionic functionalgroup-containing impurity other than the aimed compound was notdetected.

Example 6-1 Synthesis of carboxyl compound (group I(h)) (R=methyl group,A²=oxyethylene group, m=475)

Into a 1000 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were charged 410 g of the compound of the formula (p6), 450 g oftoluene, and 0.041 g of BHT, and the whole was heated under reflux at110° C. or higher to effect azeotropic removal of water. After thereaction liquid was cooled to 40° C., 20.5 g of potassium hydroxide wasadded thereto and, after 1 hour of stirring, 8.7 g of ethylbromohexanoate was added dropwise to the mixture over a period of 15minutes with maintaining it at 40° C., followed by 7.5 hours ofstirring. Furthermore, 20.5 g of potassium hydroxide was added theretoand, after 30 minutes of stirring, 8.7 g of ethyl bromohexanoate wasadded dropwise to the mixture over a period of 30 minutes withmaintaining it at 40° C., followed by 6.5 hours of stirring. To thereaction liquid was added 410 g of water for injection, and the wholewas heated to 50° C. and stirred for 2 hours. Subsequently, the reactionmixture was cooled to 10° C. or lower and 97 g of 85% phosphoric acidwas slowly added so that temperature of the reaction liquid did notexceed 10° C. To the reaction liquid were added 400 ml of ethyl acetate,400 ml of hexane, and 0.041 g of BHT, and the whole was stirred for 15minutes, followed by leaving the mixture on standing until layersseparated. After the layer separation, the upper organic layer wasremoved with suction and 600 ml of chloroform was added to the loweraqueous layer. After 15 minutes of stirring, the whole was left onstanding until layers separated. After the layer separation, the lowerchloroform layer was taken out and 400 ml of ethyl acetate and 100 g ofmagnesium sulfate were added to remove water. After the magnesiumsulfate was removed by filtration, hexane was added to the filtrateuntil crystals were precipitated. After the crystals were collected byfiltration, the crystals were dissolved under heating in 400 ml of ethylacetate. After dissolution, the solution was cooled until crystals wereprecipitated. The crystallization operation was repeated five times andthe resulting crystals were dried to obtain the compound of thefollowing (p12).

¹H-NMR (CDCl₃, internal standard: TMS) δ(ppm): 1.42 (2H, m, —OCH₂CH₂ CH² CH₂CH₂COOH), 1.63 (2H, m, —OCH₂ CH ² CH₂CH₂CH₂COOH), 1.67 (2H, m,—OCH₂CH₂CH₂ CH ² CH₂COOH), 2.80 (2H, m, —OCH₂CH₂CH₂CH₂ CH ² COOH), 3.38(6H, s, —CH ³ ), 3.40-3.80 (3807H, m, —CH ² O(CH ² CH ² O)_(m)CH₃,CHO(CH ² CH ² O)_(m)CH₃, —CH ² OCH ² CH₂CH₂CH₂CH₂COOH).

<main peak> number average molecular weight (Mn): 41632, weight averagemolecular weight (Mw): 42314, polydispersity (Mw/Mn): 1.016, peak topmolecular weight (Mp): 42579;

<whole peak> number average molecular weight (Mn): 40084, weight averagemolecular weight (Mw): 41561, polydispersity (Mw/Mn): 1.037, peak topmolecular weight (Mp): 42579.

Low-molecular-weight impurities: 3.60%

Example 6-2

When the compound of the formula (p12) was analyzed on a liquidchromatography using an ion-exchange column (concentration of ammoniumformate buffer solution: 0.15 mM), any ionic functional group-containingimpurity other than the target compound was not detected.

Example 7-1

The compound of the following (p13) having a molecular weight of about10,000 was synthesized in a similar manner to Example 1-3.

¹H-NMR (CDCl₃, internal standard: TMS) δ(ppm): 3.40-3.80 (933H, m, —CH ²O(CH ² CH ² O)_(m)H, CHO(CH ² CH ² O)_(m)H, CH ² OCH₂Ph), 4.54 (2H, s,—CH ² Ph), 7.27-7.38 (5H, m, —CH₂ Ph).

Example 7-2

The compound of the following (p14) was synthesized in a similar mannerto Example 14, by using the compound (p13) as a starting material.

¹H-NMR (CDCl₃, internal standard: TMS) δ(ppm): 3.38 (6H, s, —CH ³ ),3.40-3.80 (933H, m, —CH ² O(CH ² CH ² O)_(m)CH₃, CHO(CH ² CH ²O)_(m)CH₃, CH ² OCH₂Ph), 4.54 (2H, s, —CH ² Ph), 7.27-7.38 (5H, m, —CH₂Ph).

Example 7-3

The compound of the following (p15) was synthesized in a similar mannerto Example 1-5, by using the compound (p14) as a starting material.

¹H-NMR (CDCl₃, internal standard: TMS) δ(ppm): 3.38 (6H, s, —CH ³ ),3.40-3.80 (933H, m, —CH ² O(CH ² CH ² O)_(m)CH₃, CHO(CH ² CH ²O)_(m)CH₃, CH ² OH).

Example 8 Synthesis of aldehyde compound (group I(j)) (R=methyl group,A²O=oxyethylene group, m=116) Example 8-1

Into a 200 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were charged 20 g of the above compound (p15) and 120 g of toluene,and the whole was heated under reflux to effect azeotropic removal ofwater. After cooling to room temperature, 1.012 g of triethylamine and0.687 g of methanesulfonyl chloride were added thereto, followed by 3hours of reaction at 40° C. After filtration of the reaction liquid, thefiltrate was charged into a 500 ml beaker and crystallization wascarried out with adding 100 ml of ethyl acetate and 150 ml of hexane.The crystallization operation was repeated three times. The crystalswere collected by filtration and dried to obtain the following mesylatecompound (p16).

¹H-NMR (CDCl₃, internal standard: TMS) δ(ppm): 3.08 (3H, s, —SO₃ CH ³ ),3.38 (6H, s, —CH ³ ), 3.40-3.80 (931H, m, —CH ² O(CH ² CH ² O)_(m)CH₃,CHO(CH ² CH ² O)_(m)CH₃), 4.27-4.44 (2H, m, —CH ² OSO₃CH₃).

Example 8-2

Into a 200 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, a Dean-stark tube, and a condensertube were added 10 g of the above mesylate compound (p16), 0.001 g ofBHT, and 100 ml of toluene, and the whole was heated under reflux toeffect azeotropic removal of water, followed by cooling to roomtemperature. On the other hand, into a 100 ml round-bottom flask fittedwith a thermometer, a nitrogen-introducing tube, a stirrer, a Dean-starktube, and a condenser tube were added 14.8 g of 3,3-diethoxy-1-propanoland 40 ml of toluene, and the whole was heated under reflux to effectazeotropic removal of water. After cooling to room temperature, 0.36 gof sodium was added and the whole was stirred at room temperature for 2hours until it was dissolved. After dissolution of the sodium wasconfirmed, the reaction liquid was poured into the round-bottom flaskcontaining the compound (p16) from which water had been removed asabove, followed by 4 hours of reaction at 70° C. After cooling of thereaction liquid to 40° C., 0.36 g of ion-exchange water was added andthe whole was stirred for 30 minutes, followed by filtration. Then, 100ml of ethanol was added to the filtrate, and hexane was added untilcrystals were precipitated. The crystallization operation was repeatedfive times. The resulting crystals were collected by filtration anddried to obtain the following acetal compound (p17).

¹H-NMR (CDCl₃, internal standard: TMS) δ(ppm): 1.20 (6H, t,—CH₂CH₂CH(OCH₂ CH ³ )₂), 1.88-1.92 (2H, m, —CH₂ CH ² CH(OCH₂CH₃)₂), 3.38(6H, s, —CH ³ ), 3.40-3.80 (939H, m, —CH ² O(CH ² CH ² O)_(m)CH₃, CHO(CH² CH ² O)_(m)CH₃, —CH ² O—CH ² CH₂CH(OCH ² CH₃)₂), 4.64 (1H, t, —CH₂CH₂CH(OCH₂CH₃)₂).

Example 8-3

Into a 200 ml beaker was weighed 4 g of the resulting acetal compound(p17). Then, 80 g of ion-exchange water was added to dissolve thecrystals and the solution was adjusted to pH 1.5 with 85% phosphoricacid, followed by 2 hours of stirring at room temperature. During thereaction, light shielding was conducted. Thereafter, 16 g of sodiumchloride was added and dissolved and the whole was adjusted to pH 7.0with 40% aqueous sodium hydroxide solution. To the reaction liquid wasadded 30 ml of chloroform, and the whole was stirred for 15 minutes,followed by leaving the mixture on standing until layers separated. Thelower chloroform layer was dried over sodium sulfate and, afterfiltration, 50 ml of toluene and 120 ml of hexane was added toprecipitate crystals. The crystals was collected by filtration and driedto obtain the following aldehyde compound (p18).

¹H-NMR (CDCl₃, internal standard: TMS) δ(ppm): 2.65 (2H, m, CH ² COH),3.38 (6H, s, —CH ³ ), 3.40-3.80 (935H, m, —CH ² O(CH ² CH ² O)_(m)CH₃,CHO(CH ² CH ² O)_(m)CH₃, CH ² OCH ² CH₂COH), 9.78 (1H, m, CH₂COH).

<main peak> number average molecular weight (Mn): 10055, weight averagemolecular weight (Mw): 10213, polydispersity (Mw/Mn): 1.016, peak topmolecular weight (Mp): 10441;

<whole peak> number average molecular weight (Mn): 9779, weight averagemolecular weight (Mw): 10252, polydispersity (Mw/Mn): 1.049, peak topmolecular weight (Mp): 10441.

Low-molecular-weight impurities: 3.50%.

Example 9

For evaluating stability of the compounds of the invention, thefollowing model compound was synthesized and the stability was compared.

Example 9-1

In 50 ml of methanol was dissolved 63 mg (20 mM) of sodiumcyanotrihydroborate. Into 2 ml of the solution were added 0.5 g of thealdehyde compound (p 18) and 50 μl of n-butylamine, followed by 18 hoursof stirring at room temperature. Methanol was removed by evaporation toeffect concentration and then the concentrate was extracted by adding 20ml of chloroform and 20 ml of 20% aqueous sodium chloride. Theextraction operation was repeated three times. The resulting chloroformlayer was dried over sodium sulfate and, after filtration, concentrated.The resulting concentrate was dissolved under heating by adding 20 ml ofethyl acetate and then 30 ml of hexane was added to precipitatecrystals, which was collected by filtration. The resulting crystals wereweighed into a 100 ml beaker and dissolved under heating with adding 20ml of ethyl acetate and then 20 ml of hexane was added to precipitatecrystals again, which was collected by filtration and dried to obtainthe following compound (p19).

Example 9-2 Evaluation of Stability (Accelerated Aging Test)

The synthesized above compound (p19) was weighed in an amount of 12 mg,and 1 ml of 100 mM phosphate buffer solution (pH=8.8) was added thereto,followed by 12 hours of stirring on a water bath at 75° C. GPCmeasurement was carried out before starting and after completion ofstirring. The results are shown in FIG. 4 and FIG. 5. FIG. 4 is a GPCchart of the sample of the compound (p19) before starting and FIG. 5 isa GPC chart of the sample of(p19) after heating.

Comparative Example 1

The following compound (p20) having a molecular weight of about 10700purchased from Shearwater Polymers, Inc. was weighed in an amount of 107mg, and 10 μl of n-butylamine and 1 ml of chloroform were added thereto,followed by 18 hours of stirring at room temperature. Chloroform wasremoved by evaporation to effect concentration, then the concentrate wasdissolved under heating with adding 20 ml of ethyl acetate, and then 30ml of hexane was added to precipitate crystals, which was collected byfiltration. The resulting crystals were weighed into a 100 ml beaker anddissolved under heating with adding 20 ml of ethyl acetate, and then 20ml of hexane was added to precipitate crystals again, which wascollected by filtration and dried to obtain the following compound(p21).

Using the above synthesized compound (p21), the same operations as inExample 9-2 were carried out and GPC measurement was conducted. Theresults are shown in FIG. 6 and FIG. 7. FIG. 6 is a GPC chart of thesample of the compound (p21) before starting and FIG. 7 is a GPC chartof the sample of (p21) after heating.

From the results of FIG. 4 and FIG. 5, it was revealed that thecompounds of the invention were not hydrolyzed and exhibited a highstability. On the other hand, from the results of FIG. 6 and FIG. 7, acompound having a molecular weight of 1/2 was formed in an amount ofabout 25% in the case of (p21) of Comparative Example, which showed thatthe urethane bond was cleaved and the branched polyethylene glycol wasdecomposed into a single chain.

Example 10 Synthesis of succinimide ester compound (group I(c))(R=methyl group, A²O=oxyethylene group, m=475)

Into a 200 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, and a condenser tube were charged10 g of the carboxyl compound of the formula (p12), 50 ml of drytoluene, 0.01 g of BHT, and 0.1 g of sodium acetate, and the whole washeated to dissolved them. The reaction liquid was heated to 50° C. and55 mg of N-hydroxysuccinimide was added thereto. After 1 hour ofstirring, the reaction liquid was cooled to 40° C. After cooling, 98 mgof DCC was added thereto, followed by 4 hours of reaction. Aftercompletion of the reaction, the reaction liquid was filtered to removeDCU. Then, 50 ml of hexane was added to the filtrate to precipitatecrystals and the crystals were collected by filtration. The collectedcrystals were dissolved under heating with adding 20 ml of acetonitnrleand 200 ml of ethyl acetate. Then, 200 ml of hexane was added thereto toprecipitate crystals and the crystals were collected by filtration. Thecrystallization operation was repeated three times and the resultingcrystals were dried to obtain the compound of the following (p22).

¹H-NMR (CDCl₃, internal standard: TMS) δ(ppm): 1.46 (2H, m, —OCH₂CH₂ CH² CH₂CH₂COON), 1.58 (2H, m, —OCH₂ CH ² CH₂CH₂CH₂COON—), 1.77 (2H, m,—OCH₂CH₂CH₂ CH ² CH₂COON), 2.61 (2H, m, —OCH₂CH₂CH₂CH₂ CH ² COON), 2.84(4H, s, —NHS), 3.38 (6H, s, —CH ³ ), 3.40-3.80 (3807H, m, —CH ² O(CH ²CH ² O)_(m)CH₃, CHO(CH ² CH ² O)_(m)CH₃, —CH ² —OCH ² CH₂CH₂CH₂CH₂COOH).

<main peak> number average molecular weight (Mn): 41632, weight averagemolecular weight (Mw): 42314, polydispersity (Mw/Mn): 1.016, peak topmolecular weight (Mp): 42579;

<whole peak> number average molecular weight (Mm): 40084, weight averagemolecular weight (Mw): 41561, polydispersity (Mw/Mn): 1.037, peak topmolecular weight (Mp): 42579.

Low-molecular-weight impurities: 3.60%

Example 11 Synthesis of succinimide ester compound (group I(c))(R=methylgroup, A²O=oxyethylene group, m=223)

Into a 500 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, a stirrer, and a condenser tube were charged50 g (2.5 mmol) of the compound of the formula (p3), 250 ml of toluene,0.05 g of BHT, and 0.5 g of sodium acetate, and the whole was heatedunder reflux to effect azeotropic removal of water. To the reactionliquid was added 2.853 g of glutaric anhydride, followed by 15 hours ofreaction at 110° C. Thereafter, the reaction liquid was cooled to 40° C.and 6.905 g of N-hydroxysuccinimide was added thereto. After 1 hour ofstirring, 10.317 g of DCC was added thereto, followed by 5 hours ofreaction at 40° C. After completion of the reaction, the reaction liquidwas filtered to remove DCU. Then, 250 ml of hexane was added to thefiltrate to precipitate crystals and the crystals were collected byfiltration. The collected crystals were dissolved under heating withadding 25 ml of acetonitrile and 250 ml of ethyl acetate. Then, 250 mlof hexane was added thereto to precipitate crystals and the crystalswere collected by filtration. The crystallization operation was repeatedfive times and the resulting crystals were dried to obtain the compoundof the following (p23).

¹H-NMR (CDCl₃, internal standard: TMS) δ(ppm): 2.05 (2H, t, —OCOCH₂ CH ²CH₂COON), 2.49 (2H, t, —OCOCH ² CH₂CH₂COON—), 2.71 (2H, t, —OCOCH₂CH₂ CH² COON), 2.84 (4H, s, succinimide), 3.38 (6H, s, —CH ³ ), 3.40-3.80(178H, m, —CH ² O(CH ² CH ² O)_(m)CH₃, CHO(CH ² CH ² O)_(m)CH₃).4.11-4.15, 4.26-4.30 (1H, 1H, m, m, CH ² —OCOCH₂CH₂CH₂COON—)

<main peak> number average molecular weight (Mn): 19883, weight averagemolecular weight (Mw): 20217, polydispersity (Mw/Mn): 1.017, peak topmolecular weight (Mp): 20162;

<whole peak> number average molecular weight (Mn): 19503, weight averagemolecular weight (Mw): 20087, polydispersity (Mw/Mn): 1.030, peak topmolecular weight (Mp): 20162.

Low-molecular-weight impurities: 1.04%

Comparative Example 2-1

To a 300 ml round-bottom flask fitted with a thermometer, anitrogen-introducing tube, and a stirrer were added 27.3 g (0.15 mol) of3-benzyloxy-1,2-propanediol, 135 g of dry toluene, and 0.9 g (39 mmol:26% by mol) of sodium. With introducing nitrogen thereinto, the wholewas stirred at 80° C. until sodium dissolved. After dissolution, thesolution was further stirred at 80° C. for 2 hours.

The reaction liquid was charged into a 5 L autoclave thoroughly driedbeforehand and the same operations as in Examples 1-3, 1-6, 1-7, and 1-8were conducted to obtain the compound (p24) having the same structure asthat of (p6).

Comparative Example 2-2

The compound of the following (p25) having the same structure as that of(p9) was synthesized in a similar manner to Examples 2-1, 2-2, and 3-1,by using the compound (p24) as a starting material.

<main peak> number average molecular weight (Mn): 40456, weight averagemolecular weight (Mw): 41857, polydispersity (Mw/Mn): 1.043, peak topmolecular weight (Mp): 42700;

<whole peak> number average molecular weight (Mn): 32878, weight averagemolecular weight (Mw): 37658, polydispersity (Mw/Mn): 1.099, peak topmolecular weight (Mp): 42700.

Low-molecular-weight impurities: 19.12%

Example 12

Twenty miligrams of the compound (p25) was dissolved in deuteratedmethanol. The solution was measured on ¹H-NMR (number of integrationtimes: 256) and M2/(M1+M2)×100 was calculated. The results are shown inTable 3 together with those of (p9). TABLE 3 M1 M2 M2/(M1 + M2) × 100(p9) 2 0.0170 0.84 (p25) 2 0.6606 24.83

Example 13 Modification of Peptide

A peptide of Humanin(Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr-Ser-Glu-Ile-Asp-Leu-Pro-Val-Lys-Arg-Arg-Ala)(molecular weight: 2687.2) was adjusted to 0.5 μM with 10 mM phosphatebuffer (pH=6.4). Into 200 μl of the solution was added 4 mg of thecompound of the formula (p9) or (p25), followed by 4 hours of reactionat room temperature. Then, 200 μl of the reaction liquid was chargedinto an SP-Sepharose FF (manufactured by Amersham) column, which wasthen equilibrated with 20 mM Tris-HCl buffer (pH=8.2). After theequilibration, a solution obtained by adding NaCl to the buffer so as tobe 1N was passed through the column and a fraction of the peptidemodified with (p9) or (p25) was obtained with monitoring the elute byUV. Thereafter, 20 μl of the fraction was mixed with 20 μl of a Tris-SDSsample-treating liquid, followed by heating on a boiling water bath for2 minutes and 30 seconds. Then, 20 μl of the solution was analyzed bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (4-20%). Thestaining was carried out by BaI₂ staining. The results were shown inFIG. 8. The left lane is a result of modification with (p9) and theright lane is a result of modification with (p25). As a result, it wasfound that the compound having a polydispersity of more than 1.07 and alarge value of M2/(M1+M2)×100 has a broad band of a peptide and thushomogeneous modification was not achieved.

1. A polyalkylene glycol derivative comprising a compound of the formula(1):

wherein R is a hydrocarbon group having 1 to 24 carbon atoms, OA² is anoxyalkylene group having 2 to 4 carbon atoms, the groups represented byR are the same or different from each other in one molecule, the groupsrepresented by OA² are the same or different from each other in onemolecule, m is an average number of moles of the above oxyalkylene groupadded, m represents 10 to 1000, and X represents a functional groupcapable of chemically reacting with a bio-related substance,polydispersity Mw/Mn of the polyalkylene glycol derivative in gelpermeation chromatography satisfying the following relationship:Mw/Mn≦1.07 wherein Mw represents a weight average molecular weight andMn represents a number average molecular weight.
 2. The polyalkyleneglycol derivative according to claim 1, comprising low-molecular-weightimpurities in an amount of 6% or less in gel permeation chromatography.3. The polyalkylene glycol derivative according to claim 2, wherein R isa hydrocarbon group having 1 to 10 carbon atoms, OA² is an oxyalkylenegroup having 2 to 3 carbon atoms, and m is 20 to 1000 in the formula(1).
 4. The polyalkylene glycol derivative according to claim 2, whereinR is a methyl group, OA² is an oxyethylene group, and m is 50 to 1000 inthe formula (1).
 5. A modified bio-related substance obtainable byreacting a bio-related substance with the polyalkylene glycol derivativeaccording to claim
 2. 6. The polyalkylene glycol derivative according toclaim 2, wherein X is a group selected from the group (I):

wherein Z represents an alkylene group alone or an alkylene groupcontaining an ether bond, an ester bond, a urethane bone, an amide bond,a carbonate bond, or a secondary amino group.
 7. The polyalkylene glycolderivative according to claim 2, wherein X is a group represented by theformula (a):-Z-NH₂   (a) wherein Z represents an alkylene group alone or an alkylenegroup containing an ether bond, an ester bond, a urethane bond, an amidebond, a carbonate bond, or a secondary amino group.
 8. The polyalkyleneglycol derivative according to claim 2, wherein X is a group representedby the formula (b):

wherein Z represents an alkylene group alone or an alkylene groupcontaining an ether bond, an ester bond, a urethane bond, an amide bond,a carbonate bond, or a secondary amino group.
 9. The polyalkylene glycolderivative according to claim 2, wherein X is a group represented by theformula (c):

wherein Z represents an alkylene group alone or an alkylene groupcontaining an ether bond, an ester bond, a urethane bond, an amide bond,a carbonate bond, or a secondary amino group.
 10. The polyalkyleneglycol derivative according to claim 2, wherein X is a group representedby the formula (d):


11. The polyalkylene glycol derivative according to claim 2, wherein Xis a group represented by the formula (e):

wherein Z represents an alkylene group alone or an alkylene groupcontaining an ether bond, an ester bond, a urethane bond, an amide bond,a carbonate bond, or a secondary amino group and W¹ is a halogen atomselected from Cl, Br, and I.
 12. The polyalkylene glycol derivativeaccording to claim 2, wherein X is a group represented by the formula(f):

wherein Z represents an alkylene group alone or an alkylene groupcontaining an ether bond, an ester bond, a urethane bond, an amide bond,a carbonate bond, or a secondary amino group.
 13. The polyalkyleneglycol derivative according to claim 2, wherein X is a group representedby the formula (g):-Z-ONH₂   (g) wherein Z represents an alkylene group alone or analkylene group containing an ether bond, an ester bond, a urethane bond,an amide bond, a carbonate bond, or a secondary amino group.
 14. Thepolyalkylene glycol derivative according to claim 2, wherein X is agroup represented by the formula (h):-Z-COOH   (h) wherein Z represents an alkylene group alone or analkylene group containing an ether bond, an ester bond, a urethane bond,an amide bond, a carbonate bond, or a secondary amino group.
 15. Thepolyalkylene glycol derivative according to claim 2, wherein X is agroup represented by the formula (i):-Z-SH   (i) wherein Z represents an alkylene group alone or an alkylenegroup containing an ether bond, an ester bond, a urethane bond, an amidebond, a carbonate bond, or a secondary amino group.
 16. The polyalkyleneglycol derivative according to claim 2, wherein X is a group representedby the formula (j):

wherein Z represents an alkylene group alone or an alkylene groupcontaining an ether bond, an ester bond, a urethane bond, an amide bond,a carbonate bond, or a secondary amino group.
 17. The polyalkyleneglycol derivative according to claim 7, wherein an amount of impuritiescontaining an ionic functional group other than a target product is 1%or less in a chromatogram obtained by analyzing a polyalkylene glycolderivative represented by the formula (1) by liquid chromatography usingan ion-exchange column.
 18. The polyalkylene glycol derivative accordingto claim 14, wherein an amount of impurities containing an ionicfunctional group other than a target product is 1% or less in achromatogram obtained by analyzing a polyalkylene glycol derivativerepresented by the formula (1) by liquid chromatography using anion-exchange column.
 19. The polyalkylene glycol derivative according toclaim 8, wherein an amount of impurities containing an ionic functionalgroup other than an objective product is 1% or less in a chromatogramobtained by reacting a polyalkylene glycol derivative represented by theformula (1) with a labeling agent having an ionic functional group andanalyzing the resulting product by liquid chromatography using anion-exchange column.
 20. The polyalkylene glycol derivative according toclaim 10, wherein an amount of impurities containing an ionic functionalgroup other than an objective product is 1% or less in a chromatogramobtained by reacting a polyalkylene glycol derivative represented by theformula (1) with a labeling agent having an ionic functional group andanalyzing the resulting product by liquid chromatography using anion-exchange column.
 21. The polyalkylene glycol derivative according toclaim 8, which satisfies the following parameter:M2/(M1+M2)×100≦2 M1: an integral value of the maleimido group derivedfrom the hydroxyl group at the 1-position directly bonded to theglycerin skeleton when a nuclear magnetic resonance spectrum of apolyalkylene glycol derivative represented by the formula (1) is obtainin deuterated methanol; M2: an integral value of the maleimido groupderived from the hydroxyl group of the polyalkylene glycol chain. 22.The polyalkylene glycol derivative according to claim 19, whichsatisfies the following parameter:M2/(M1+M2)×100≦2 M1: an integral value of the maleimido group derivedfrom the hydroxyl group at the 1-position directly bonded to theglycerin skeleton when a nuclear magnetic resonance spectrum of apolyalkylene glycol derivative represented by the formula (1) is obtainin deuterated methanol; M2: an integral value of the maleimido groupderived from the hydroxyl group of the polyalkylene glycol chain.